Quadrupole ion trap mass spectrometry of peptides - CaltechTHESIS
CaltechTHESIS
A Caltech Library Service
About
Browse
Deposit an Item
Instructions for Students
Quadrupole ion trap mass spectrometry of peptides
Citation
Jonscher, Karen Rae
(1997)
Quadrupole ion trap mass spectrometry of peptides.
Dissertation (Ph.D.), California Institute of Technology.
doi:10.7907/9btc-bk47.
Abstract
Biological mass spectrometry addresses the challenging unsolved structural issues surrounding biopolymers of fundamental importance to the biomedical sciences. Key to this discipline is the ability to extract useful information from complex peptide mixtures. Several approaches were developed to analyze peptides utilizing the unique capabilities of the quadrupole ion trap mass spectrometer. An external matrix-assisted laser desorption ionization source was constructed. Detection of peptides in the mid-femtomole range and of proteins in the low-femtomole range was reported. Singly-charged molecules with molecular weights in excess of 34,000 u were observed. Peptides generated by enzymatic digestion of the P protein of Sendai virus were separated by HPLC and the technique was successfully applied to locate phosphorylation sites.
A hybrid quadrupole mass filter/quadrupole ion trap mass spectrometer was assembled. Peptide mixtures were separated by sequentially transmitting one value of m/z into the ion trap for mass analysis. The sequential injection technique served to significantly reduce space charge-induced suppression effects and improved resolution and fragmentation efficiency when compared to results obtained using an ion trap. A novel method of scanning afforded the ability to perform neutral loss experiments for the identification of phosphopeptides in a mixture. A long duty cycle, due to acquisition hardware, limited the utility of this approach for continuous ionization techniques.
A low flowrate ionization source was constructed and interfaced to the hybrid and to an ion trap. A unique needle configuration provided a detection limit of 75 attomole of a peptide mixture infused into the source. A new type of liquid junction was developed to apply voltage to the sample consisting of a platinum wire inserted into the sidewall of a length of Teflon tubing. The junction was versatile, robust, and easy to use and performance compared well with other types of junctions. Capillary electrophoresis and hydrophobic membranes were used to separate peptide mixtures. Detection limits of the techniques were 1 femtomole and 10 femtomoles, respectively, for angiotensin. Differential release of peptides using step elutions from the hydrophobic membrane was demonstrated, providing a sensitive, high throughput means of mixture simplification prior to separation by capillary electrophoresis.
Item Type:
Thesis (Dissertation (Ph.D.))
Degree Grantor:
California Institute of Technology
Division:
Engineering and Applied Science
Major Option:
Applied Physics
Thesis Availability:
Public (worldwide access)
Research Advisor(s):
Hood, Leroy E. (advisor)
Yates, John (advisor)
Thesis Committee:
Unknown, Unknown
Defense Date:
30 September 1996
Record Number:
CaltechETD:etd-01142008-075423
Persistent URL:
DOI:
10.7907/9btc-bk47
Default Usage Policy:
No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:
170
Collection:
CaltechTHESIS
Deposited By:
Imported from ETD-db
Deposited On:
28 Jan 2008
Last Modified:
16 Apr 2021 22:18
Thesis Files
Preview
PDF (Jonscher_kr_1997.pdf)
- Final Version
See Usage Policy.
8MB
Repository Staff Only:
item control page
CaltechTHESIS is powered by
EPrints 3.3
which is developed by the
School of Electronics and Computer Science
at the University of Southampton.
More information and software credits
QUADRUPOLE ION TRAP MASS
SPECTROMETRY OF PEPTIDES
Thesis by
Karen R. Jonscher
In Partial Fulfillment of the Requirements
for the Degree of
Doctor of Philosophy
California Institute of Technology
Pasadena, California
1997
(Submitted September 30, 1996)
To Peter, Spencer, and Raleigh and in loving memory of my grandmother, Sally Fried.
Acknowledgments
I feel grateful that my mentors, Dr. Lee Hood and Dr. John Yates, provided me
with a supportive atmosphere throughout my graduate career. I have been fortunate to
have 2.8 children and continue my graduate work. Dr. Hood's vision and enthusiasm
excited my imagination and sparked my interest in molecular biology. Dr. John Yates
provided insightful comments and helpful discussions. He has made the last few years
challenging and stimulating and as a result, I have developed a keen interest in the field of
biological mass spectrometry.
I have had a great deal of help from a number of people over the past several years.
Dr. Jae Schwartz built the ion trap mass spectrometer that was used for most of the work
presented in this dissertation. His technical expertise was much appreciated. Jon DeGnore
and Richard Yost from the University of Florida provided the wonderful ion trap.
renderings found in Chapters One and Two. These can be accessed on their web page at
graciously provided by Dr. K.C. Gupta at Rush Presbyterian St. Luke's Medical Center,
Chicago, IL. Bill Loyd gave unstintingly of his technical expertise and his friendship. The
guys in the machine shop: Tom, Eric, Brian, Doug, and Norm were incredibly helpful with
‘designs and often manufactured things for me while I waited. The people in our lab
provided helpful discussions in addition to their friendship. Thank you to Dave, Andy,
Lara, Edwin, Ashok and Tina. A special word of thanks to Dr. Ashley McCormack who
shared her mass spectrometry experience and friendship. In addition, she was an excellent
editor.
iv
Finally, I would like to thank my family. My parents and in-laws have been
rooting for me throughout. My husband, Peter, should be nominated for sainthood. My
children Spencer (age 4), Raleigh (age 2) and 7??? (due 11/11/96) have made it all
worthwhile. Their unconditional love has helped me to believe in myself.
Abstract
Biological mass spectrometry addresses the challenging unsolved structural issues
surrounding biopolymers of fundamental importance to the biomedical sciences. Key to
this discipline is the ability to extract useful information from complex peptide mixtures.
Several approaches were developed to analyze peptides utilizing the unique capabilities of
the quadrupole ion trap mass spectrometer. An external matrix-assisted laser desorption
ionization source was constructed. Detection of peptides in the mid-femtomole range and
of proteins in the low-femtomole range was reported. Singly-charged molecules with
molecular weights in excess of 34,000 u were observed. Peptides generated by enzymatic
digestion of the P protein of Sendai virus were separated by HPLC and the technique was
successfully applied to locate phosphorylation sites.
A hybrid quadrupole mass filter/quadrupole ion trap mass spectrometer was
assembled. Peptide mixtures were separated by sequentially transmitting one value of m/z
into the ion trap for mass analysis. The sequential injection technique served to
significantly reduce space charge-induced suppression effects and improved resolution and
fragmentation efficiency when compared to results obtained using an ion trap. A novel
method of scanning afforded the ability to perform neutral loss experiments for the
identification of phosphopeptides in a mixture. A long duty cycle, due to acquisition
hardware, limited the utility of this approach for continuous ionization techniques.
A low flowrate ionization source was constructed and interfaced to the hybrid and
to an ion trap. A unique needle configuration provided a detection limit of 75 attomole of a
peptide mixture infused into the source. A new type of liquid junction was developed to
apply voltage to the sample consisting of a platinum wire inserted into the sidewall of a
length of Teflon tubing. The junction was versatile, robust, and easy to use and
performance compared well with other types of junctions. Capillary electrophoresis and
hydrophobic membranes were used to separate peptide mixtures. Detection limits of the
techniques were 1 femtomole and 10 femtomoles, respectively, for angiotensin.
Differential release of peptides using step elutions from the hydrophobic membrane was
demonstrated, providing a sensitive, high throughput means of mixture simplification prior
to separation by capillary electrophoresis.
Table of Contents
Abstract Vv
Table of Contents vii
List of Tables and Schemes x
List of Figures xi
Chapter 1: Introduction 1
1.1 Thesis OVErvView........ccsescsssessseseesesscsssssssessesssssesesssssssscssesscssssssssseseseucasesucseenseness 1
1.2 Role of Mass Spectrometry in Biological Research..........c.cccccesescsscssesecscsscssceaeeees 2
1.3 History of the Development of Ion Traps............ccccsccsssssscsssssessssssescescssserscesscsesees 3
1.4 Strategies for Protein Sequencing by Tandem Mass Spectrometry..........0.ccccce00 8
1.4.1 Classical Microsequencing Techniques .............cccccsscsscsssesssscecceseeccescssessceseenes 8
1.4.2 Protein Sequencing By Tandem Mass Spectrometry ...........cccccccesseceseeeeeesees 9
1.4.3 Ionization Techniques for Biomolecules..............:cccccccscssssessscseccescsseeseseeseeeees 9
1.4.3.1 Ionization By Atom/Ion Bombardment..............cccccscsscssseseessesccseeseeees 9
1.4.3.2 Matrix-Assisted Laser Desorption Tonization.........ccccceeceesessessesscesees 11
1.4.3.3 Electrospray Tomization ............eccecseescssssseseessssecsessscssessssscsscsseasenseneeeces 12
1.4.4 Sample Preparation... ssssssecssseeeessecessesssessssesssseseesecaseussssavsecsesecaseases 13
1.4.5 Fragmentation of Peptides in Low Energy CID Processes ...........:cccscsesesee: 13
1.4.6 Data Interpretation 0... tcssesseseeeesessessssesessesesssscessesssssscsssacaceassessesseaeeaees 14
1.4.7 Analysis of MS Data Using Known Sequences............cccccccccssscseceessesscesecees 18
1.4.7.1 Peptide Mass Mapping..............ccccsssssssssesesssesaseccsscssssssscsceseaseeseceeeeaes 18
1.4.7.2 Computer-Aided Interpretation of Fragmentation Mass Spectra......... 19
1.4.7.3 de novo Computer Interpretation ............cccccsscssssscsssesveeseceesstscssceceerens 20
1.5 COnClUSION..........ccccesesesssssseesstssessseseseseneseseaes acetenarenensensenseassassasessessosssenssessessesseses 21
1.6 References................ ssssseseacsesussssesececueseassesesesseusssssssacassuesssesesesessscsessarscseavacecaeaeasees 22
Chapter 2: Practical Aspects of Ion Trap Theory 28
2.1 Theoretical OVErVieW..........cseeeesssesecessssessssesscasetesessssssssessssecsssscssesscatcesseesersaceaeass 28
2.2 Practical Aspects of Ion Trap Theory ...........csccsesssesessssesssscsscsscsusscscssssecaceeeeesasers 37
2.2.1 Ton Injection... cece eeeseseeeeesescesessesssesescescssesecsecsucssssscsscsessceaccaseacensensacs 40
2.2.2 lon Trapping............. a aetaeeaaeaseessecsaeeseeeaeacsecesacssecnsesesssesenaeessessceesssesetenstensaees 40
2.2.3 TON Ejection oo... ccc sssssssssscesscescseeccesecesessessesescssesssesssscsssesssvsseasceaseessasacsuees 44
2.2.4 Ton [solation..... ee eesesesscesseeccssesseesecsseseesssssssssssecsscssesesscescecaessseesesuseass 49
2.2.5 Tom Dissociation... sescsesseeesssssessessessesessescsesscssssscssssscssecceasesecsecssensenasace 49
2.2.6 High Resolution... scssessescsceccssssesssseesssesssscessecsesscessscssesssaseseasceesssansaens 53
2.3 Comparison with Other Methods .............ccescsssssssessssssesesssesseessssssscsssaseeseeseeseseueacs 56
2.4 The New Generation of Ion Traps..........ccscescssssssssscssesescssssssssssssssecsesacssssesscscsaseees 57
2.5 COnclusion..........ccccesceeeeeees feeaseeaseenaeenateceseesseecucsesesesseeescesetesseasecesseseneesecseesonsaeoes 59
2.6 Reference .........ccccccscscesssssssesevesseccsssesessesessusesesseseessssssesseessaussessessecesetaceseeesseccece 61
Chapter 3: Matrix-Assisted Laser Desorption of Peptides and Proteins on a
Quadrupole Ion Trap Mass Spectrometer 65
3.1 OVErVICW .......eeeeccceeteeceseecesereceeseeesenneees eeessaceeessaeeesssaesessaseceseseeeseeesesseesesseeeeseenaters 65
3.2 Instrument Development ............ ce esssessesecsssesssesescsesecessseeseecsseesssesesessesenseesseenes 66
3.2.1 Tmtroduction 0... eee sesssecssesseseseaecseeseeesceaeseeeseeseesseesaseeseseeeseeeeeeseeeeeesesenens 66
3.2.2 Experimental o00..... ee ccceseesccssceeseeeeseaseseeceseeessneesaceseeeceesesesesseeesssetsenseesneees ..68
3.2.2.1 Tomization SOUL CE... eescesseesesceesseseseeetseeetsseeeesaceeeeseecessesensecesneecees 68
3.2.2.2 Mass Spectrometry........csccsecscssessseesssessssseeseesesssessseesseesesseseeessneseneees 70
3.2.2.3 Sample Preparation............cccsessseseceseseesesccseeeseeeeseecseeesseseeseesecseeenes 71
3.2.3 Results and DisCusSion............cccsesssccsscseseesseseeseeeseacesssceeceseeeesssesetceesseeeeeess 74
3.2.3.1 Tandem Mass Spectrometry.........c:ccccsescssscessssescesssccescsscsecsseseeesecsseevers 80
3.2.3.2 Trapping large protein ions: effect of ionization exclusion limit......... 83
3.2.3.3 Detection Limits 00... eccesecsccscesescsecseeeseessecesnseessneeseeseesensesesseees 91
3.3 Application of MALDI/ITMS for Analysis of Phosphopeptides.............. cess 94
3.3.1 OVOLVICW ooo... eeseeseeeseccesseeeneeseseeesseeesceeeseesesaseeaecesecceseteenaeeeseaeeesseerens seteeeeeeeees 94
3.3.2 Introduction oo... eeeseeseeeeeeeeeeseneeeseees seeeseveeeesseessseseceeaeeeesseessseesenesesseeses OA
3.3.3 Experimental .0.........ccesescsssescseseseesseseseesseesseeseesseesneseseessseeesseeseneeseseseeseseoees 96
3.3.3.1 Mass Spectrometry........cecccceccsscecccsscsecssecseeceeesseessecesessseeseseeseaeeseees 96
3.3.3.2 Sample Preparation............cssescscssecseesecsccsseseeeessseceseessaeceatesseeeneeeeees 98
3.3.3.2.1 Chromatography.......ccececcsesscssscssesscseesssseseeseseesseseeseserseetereneoes 98
3.3.3.2.2 Edman Degradation... eessssseseseeereees vesseseeeseeeaseeeeescaseneeees 99
3.3.3.2.3 MALDI ...... cee csceeecssesecsseeeesesesesesseesseesseeesseesseeseneesseseeeseneees 99
3.3.3.3 Data AnalySis 0.0... ceccssseseseeseceeceeeessesecseessesaeereseneteeeseeesaeseeseesanees 100
3.3.4 Results and Discussion.........ccccesssesseseecsseseseeesesceteseeeeeseteeeeeseseeseaes sevenees 101
3.3.4.1 Peptide Mapping... ec eeesssseceeeeesesesecsseseseseceeeneseeeseeseneseeseesensees 101
3.3.4.2 Trypsin Digestion... cceccescsesssessccssssseessssesseceesessesesseeseseseneesenees 104
3.3.4.2.1 MALDI/Time-of-Flight Mass Spectrometry............scsseseesees 104
3.3.4.2.2 MALDI/Ion Trap Mass Spectrometry.............cessesessereeereessees 104
3.3.4.3 Chymotrypsin Digestion ........ cc esccssssssesssesseeesessessecsssseeesseseeseeeeeees 112
3.3.4.3.1 MALDI/Time-of-Flight Mass Spectrometry, Fraction #13......112
3.3.4.3.2 MALDI/Ion Trap Mass Spectrometry, Fraction #13................ 112
3.3.4.3.3 MALDI/Time-of-Flight Mass Spectrometry, Fraction #25......119
3.3.4.3.4 MALDI/Ion Trap Mass Spectrometry, Fraction #25.............. 122
Fae Po 0) 1 (02 105 0) ee 128
3.4 Reference .......ccecscesceeseseeessseceesneseessceeeeseesssseeessesceesseesesseceeesseeeeeseessesseseesensees 130
Chapter 4: Mixture Analysis Using a Quadrupole Mass Filter/Quadrupole Ion
Trap Mass Spectrometer... 135
4.1 OVErVICW......cecceesececceesessceseeceeeesseseseseseseeeseseaneseseessenseseeccessssseacessaeceseetenesteseaeesene® 135
4.2 Introduction ...........eeeseeeseees Laceevsceeessseesssaeensesneesesaeesenacseseseeeeseacesesessaecesssseesecanees 136
4.3 Experimental 00... ecessssscssescsssssesssscsscssesecssssssesssesscsccesesesecsecsaseeeseassceeseeseseneees 138
4.3.1 Ton SOULE... ee eee eeeeseseseeesseceseeecsseesscssseecesesassesorsesecssassnessarscesensesenseeeneees 141
4.3.2 Mass AnalyZers..........cecee peeedecessnesenesesseseseeccecsecsaseeedsseaeesescesensneseeeseeeeees 141
4.3.3 SymChronization .........cccecsceecessceesesecsessseeseessceseseesesesesesseeseesceeessnetseeseneeees 143
4.3.4 Sample Preparation.........ccccecsesssscscesssscssssseesssssscesesssssseseseescsenseeeseeseseasees 146
4.4 Results and Discussion.............c.ccccccssesecccsesccesesesccusscecccssecencssecccescecessccussesseeesses 147
4.4.1 Ion Injection into the Ion Trap .........ecesesssesessesesssscscssssesessecsecseescaeeseseacaes 147
4.4.2 Mass Resolution.......c.cccccccssecsssessesessessssssesscsssssssssessesececstsessesesassesecavescevssses 156
4.4.3 Tandem Mass Spectrometry..........ccccssesssssssccseseessssessssssscsecssseseces seseaseeeeees 163
AS COnCIUSION.......ccccsscsessescesescesesseceesssseesssascsassssessssecsussssscecsscssseceseesasatsaesecensaase 171
4.6 References ........ cee ssssessssescssesessesessssesssassessesesessesssavsusscsecscsassesacsccacesesesarsucsesecens 172
Chapter 5: High Sensitivity Peptide Mixture Separation Using Low-Flowrate
Electrospray Ionization 177
S.1 OVELVICW ..... ce cesessesscsscesesstecesessesessestessessesessessenscsesssssusecsessceescsecacsacssessecsucnecsessess 177
5.2 Introduction .......cceeeessessesssssssssssssessesessessessesssecsessucsscsscaseseesesarsecasesavsaeesesseseees 178
5.3 Experiment l ..0.....ccc ce sessscsssessecesssceesseseseessesacsesesssecsesssecsssssscavssecseseseacnssacatacens 181
5.3.1 Ton SOULCE oo... eeseeseecesesessessssessssesseccsesecsesscsssscscsasscesssesassccaecsssseaacaeensass 181
5.3.2 Needles and Liquid Junctions ..........ccccssssssessssesssscsssecssssssecsseceessaesscsssassacaes 184
- 3.3.2.1 Micropipette Needles with Liquid Junction .0........ccceccsscsscsseeecseeeees 185
5.3.2.2 Pulled Capillary Needles............cccccessssssssscsssssscsscscssecssccssenesacsusscesees 185
5.3.2.3 Metal Union... seseesssescsessessssssessssestesesesscsessecscseseesesacenesereueesaes 188
5.3.2.4 Teflon JUmction..... ee ecccessscssssessessssssscseceessscssssecsssacsessessacsaesaceacsares 188
93.3.3 Mass Spectrometry......cscccecscsesseseesessersssessssssssssssssonsassesesessesasssseessssenesenees 189
5.3.4 Chromatography.........ccccscscsesssssesssscscsesessssssssssesescscsesearescacscaeacacasacseasacaesens 192
5.3.4.1 Capillary Electrophoresis...........ccccccscsscesssssssccsssssssseceecsecseceseneescsssease 192
5.3.4.2 Membrane Chromatography ...........ccccccccscsccssssssssssescesecsecsscscsecsssaces 192
5.3.5 Sample Preparation...........eceesessssesessessesssessescssescsesssssssesevsesevsesacessascacuenees 194
5.4 Results and DisCussion...........ccccsscsessssesseseeesresiessssesesssessessesssesacsesseaesnsasseessseeees 197
5.4.1 Microspray Needle Development...........ccccccccssesssssssssssssssscsecseseccsscesseecseees 198
5.4.2 Liquid Junction Development............cecssessssssssesssssssessssscsesssssscsceceevsceecscscars 201
5.4.3 Separation of Peptide Mixtures... cccccssssssssesesessesesessssssssssacsvesessseaceres 203
5.4.3.1 Neutral LOSS Scam... cscsssessesesssssessssessssecsessscssssesecsessesearscesscsacanses 203
5.4.3.2 Separation by Capillary Electrophoresis ............cccccsssssssssesesesesssseseeees 204
5.4.3.3. Separation By Membrane Chromatography ...........ccccccssescesesseeseeees 213
5.4.3.3.1 Semsitivit yo... ee eesccesessesessesssessseessssesscssssssvsssecsesersetecencsscseeesaes 213
5.4.3.3.2 Mixture Simplification..........cccccsecssssssessscsssscescscessescscssssesavacs 219
S.5 COMCIUSIONL.... ec cceescssssseescececeseeeseecsessesesesassssneaesdsecsssecssssecsssesasavseversesssssesssacensees 222
5.6 References .......ccesssesessssesssesscesessecesssesscsessesseecassnesesssevsussssscsseceaseseasersessscsscsenasaes 226
Chapter 6: Summary 229
6.1 Matrix-Assisted Laser Desorption Ion Trap Mass Spectrometry ..............c.cs00+- 229
6.2 Hybrid Quadrupole Mass Filter/Quadrupole Ion Trap Mass Spectrometer......... 230
6.3 Mixture Separation by Low Flowrate Electrospray Ionization ...........cccscesessseees 231
6.4 References ........... seseveeeescenensesseatensessssssceneseueceeceenecsessenatceseessesecsseasacseaesasessenssases 232
Vita 233
List of Tables and Schemes
Table 1.1 Time line of ion trap technology development seseuneeucessseeessceseeessesettestesseeeseeenecs 6
Table 1.2 Abbreviations and incremental masses for the 20 commonly occurring amino
ACIS... .eeceesesceessesecsscnscasescescenceeeecasseescsncassssseessesenteessaeeseseeresesenss vesetessesseeseessanesseeseceneeseens 10
Scheme I Peptide fragmentation pathways...........cccccsscssssssssssescssesesesssscecesssseresssseassceneass 16
Table 5.1 Protocol for preparing CE COlMINS...........ccccccssesessessessssscscsssscscereececessceecseaes 193
Table 5.2 Dominant ions observed at 30%, 50%, and 70% methanol during step elutions
of a casein digest peptide mixture from a hydrophobic membrane..............c.ccscccsesesceseeee 223
Table 5.3 Percent relative abundance of selected ions when eluting a casein digest peptide
mixture from a hydrophobic membrane with 1:1:0.5 methanol: water:acetic acid............ 224
List of Figures
Figure 1.1 Rendering of the ion trap electrode assembly............cccccccscsssscesecccesescesesesseseaes 5
Figure 2.1 Simulation of ion trajectories in the ion trap illustrating trajectory focusing by ,
the applied rf field..:......... saseustesseesueesseeecesussscusessscsesesusosssaeessesaceeacesssessseseesesestessssesacsneesasees 30
Figure 2.2 Diagram showing regions of stability in the quadrupole ion trap parameterized
in terms of the operating voltages and frequencies............cccccscssssssssscssecssscessescesssceseasseceees 34
Figure 2.3 Values of selected working points for an ion of m/z 1500 plotted on the
Stability diagram..........cccesssssssesesessesesesssssacsessssesssesessesessssesesssssssuseessesvessssacesecsaceusassneaes 36
Figure 2.4 Experiment scan functions on the quadrupole ion trap mass spectrometer.....39
Figure 2.5 Simulation illustrating the effect of filling the ion trap with a helium damping
GAS... sccescssccsssesseesseesessessesnseaeesnenseesacseeseesessesssssseseesaeessseaesssenseseseaseaseaesstsseecseseasausessoseraseaees 42
Figure 2.6 Relative g, values for ions with three different mass-to-charge ratios............. 46
Figure 2.7 Relative g, values for ions with three different mass-to-charge ratios under
resonance ejection conditions............ seeeeeneenseeeseesasseseseneeessesessccsssecsesensessaeseneeeaecsusersneassaeeses 48
Figure 2.8 Methods of isolating a single value of m/Z in an ion trap.......ccceescesessseseees 51
Figure 2.9 Mass scan windows obtained by extending the resolution on the quadrupole
ion trap........... ceeanensensesuesnessesseensensessecneseesacessesenssssusssseseeanenesecessensaeaeeseesaueasesasenesetesescasseaseass 55
Figure 3.1 Ion trap scan functions used for matrix-assisted laser desorption ionization
EXPCTUMEMES...........ceecesssscssssssscscesesesssscsesssseseneseesesscdseessssssessestenesseassecsusssssseesatatsecaceaeaseass 73
Figure 3.2 Schematic diagram of the matrix-assisted laser desorption
ionization/quadrupole ion trap mass SpectroMetel............cccccccsscssescsscsseecsceccsecssseessarsaseseees 77
Figure 3.3 Matrix-assisted laser desorption ionization mass spectrum of 250 fmol of the
tetradecapeptide renin substrate........c.eesesessssecessessescsesesesssesesesssceceessssusecsacavavacececsasaeas 79
Figure 3.4 Matrix-assisted laser desorption ionization mass spectra of the proteins bovine
insulin and bovine CYtOCHTOME C.........eccscssscssessessesscsscssscsssssscsssccuscscsecesceecsssaseseesesseessens 82
Figure 3.5 A comparison of MS/MS spectra of the peptide angiotensin I obtained using
matrix-assisted laser desorption ionization and liquid secondary ion mass spectrometry.
The two techniques provided comparable results.............ccccccccsscsssssssessseccecccsscseesesscsncsseees 85
Figure 3.6 Graph of optimal injection rf voltage vs. the square root Of MASS..........e:eeeee 88
Figure 3.7 Matrix-assisted laser desorption ionization mass spectrum of whale myoglobin
and porcine elastase. The dimer of myoglobin is also detected..........cccccsccsessseseseseeessseees 90
Figure 3.8 Matrix-assisted laser desorption ionization mass spectrum of 10 fmol of whale
myoglobin applied to the probe tip... ee eessesessseseeeseesesesscsseeessecsscsseesssssecescasesasseceeees 93
Figure 3.9 Amino acid sequence from the P protein of Sendai virus illustrating the results
of a tryptic Mapping CXPETIMENL..... eee ecseeeeeeesecseseessesessesscsesesssessssessessssessscessasevene 103
Figure 3.10 Matrix-assisted laser desorption ionization/linear time-of-flight mass
spectrum of a phosphopeptide of m/z 2909 from the P protein of Sendai virus
corresponding to residues 255-282, YNSTGSPPGKPPSTQDEHINSGDTPAVR........106
Figure 3.11(a) Amino acid sequence of a phosphopeptide of m/z 2909 from the P protein
of Sendai virus corresponding to residues 255-282,
YNSTGSPPGKPPSTQDEHINSGDTPAVR......... ec eesccsssessscsseesssecsecessesesssssesecsascesecraee 108
Figure 3.11(b,c) Matrix-assisted laser desorption ionization/ion trap fragmentation mass
spectrum of a phosphopeptide of m/z 2909 from the P protein of Sendai virus
corresponding to residues 255-282, YNSTGSPPGKPPSTQDEHINSGDTPAVR........109
Figure 3.12 Matrix-assisted laser desorption ionization/linear time-of-flight mass
spectrum of a phosphopeptide of m/z 1728 from the P protein of Sendai virus
corresponding to residues 240-255, TPATVPGTRSPPLNRY..........c:cccccsscscssscsessessesseces 114 .
Figure 3.13(a) Amino acid sequence of a phosphopeptide of m/z 1728 from the P protein
of Sendai virus corresponding to residues 240-255, TPATVPGTRSPPLNRY.............. 117
Figure 3.13(b,c) ‘Matrix-assisted laser desorption ionization/ion trap fragmentation mass
spectrum of a phosphopeptide of m/z 1728 from the P protein of Sendai virus
Figure 3.14 Matrix-assisted laser desorption ionization/ion trap fragmentation mass
spectrum of a phosphopeptide resulting from one stage of manual Edman degradation of
the peptide of m/z 1728 from the P protein of Sendai virus corresponding to residues 240-
255, TPATVPGTRSPPLNRY........cccssscssssesceesesceeceasesssseeeassasenssseseesseesecseseeseusesseasensas 121
Figure 3.15 Matrix-assisted laser desorption ionization/linear time-of-flight mass
spectrum of a phosphopeptide of m/z 2730 from the P protein of Sendai virus
corresponding to residues 228-253, KRRPTNSGSKPLTPATVPGTRSPPLN............5. 124
Figure 3.16(a) Amino acid sequence of a phosphopeptide of m/z 2730 from the P protein
of Sendai virus corresponding to residues 228-253,
KRRPTNSGSKPLTPATVPGTRSPPLN..........cccssccssssssessssseseeeeecesescsecsesscsssecsseeserecensars 126
Figure 3.16(b) Matrix-assisted laser desorption ionization/ion trap fragmentation mass
spectrum of a phosphopeptide of m/z 2730 from the P protein of Sendai virus
corresponding to residues 228-253, KRRPTNSGSKPLTPATVPGTRSPPLN.............. 127
Figure 4.1 Diagram of the quadrupole mass filter/quadrupole ion trap mass spectrometer
(Q/QITMS).....ccccescecscssecsecsccseeseesseesessesscessesesseesseseessesassseeessessssssssecseeseeessscusssscsacseseseeeseas 140
Figure 4.2 Diagrams of scanning modes utilized on the Q/QITMS to inject ions into the
VOM CLAP... .sesecseeeeesesssseceeeseessesessssnesseseseessssesesseessecsessesseenssessenessssecsessssaesecsessesessesessssceneenensee 145
Figure 4.3 Comparison of selected ion chromatograms obtained by electron impact
ionization of perfluorotributylamine utilizing two different scanning modes of the
Q/QITMS. Sequential injection of ions into the ion trap is illustrated...........cc cece 149
Figure 4.4 Mass spectrum of the peptide Glu-fibrinopeptide B obtained by liquid SIMS
ionization. Sequential injection using the Q/QITMS was shown to reduce signal
suppression caused by Space Charge........cccsscssscseeeccseeeseesescesctseessesssseessessesseseessenseenes 153
Figure 4.5 Mass spectra and selected ion chromatograms illustrating the improvement in
spectral quality obtained by using sequential injection on the Q/QITMS for the analysis of a
simple peptide mixture. Mass chromatographic resolution was determined to be 14 u...155
Figure 4.6 High resolution liquid SIMS mass spectrum of the peptide angiotensin I
owing the ion trap acquisition scan speed by a factor Of 200............c:ccsccscessesessescsesscssssees 159
Figure 4.7 Graphical representation of a linear regression analysis of the calculated
resolution for m/z 502 obtained by electron impact of perfluorotributylamine as a function
Of COOLING TIME... ceeecescscessscesesscsssssseessesssssessssesesscseneeseeeeseacseesecseeeessesacsssusessessenesaees 162
Figure 4.8 Selected ion chromatograms illustrating automated fragmentation of ions
generated from electron impact ionization of perfluorotributylamine achieved by
decrementing the float voltage of the ion trap electrode assembly to increase the relative
INJECTION CNCTYY........ccececcssesscsesssessecececessessesseesscssseseeseeseneeseesseessessssentssseeseseaesessenssceeacs 165
Figure 4.9 Tandem mass spectra obtained for the peptide angiotensin I using the
Q/QITMS in rf-only mode and in mass selection mode illustrating improvement in
spectral quality obtained by mass selection without subsequent rf-isolation..............0000 168
Figure 5.1 Diagram of microspray ionization source and interface to Q/QITMG........... 183
Figure 5.2 Microspray needle and liquid junction configurations..............:cscscsseeeseeees 187
Figure 5.3 Diagram of the LCQ ion trap mass spectromete..............sccscssesseseesserseeseenees 191
Figure 5.4 Diagram of Teflon membrane cartridge used for separation of peptide
MIXtUTES...... ce eeeeeeeee sasesescsesevsaesecsessesesssesessesessssenssusessosseeesseeaceeesaeaessseseeaseseessenseaseatensenses 196
Figure 5.5 High sensitivity microspray infusion mass spectrum of a mixture of the
peptides angiotensin I and melittin obtained on the Q/QITMS illustrating consumption of
75 amol Of material... ee ceeescesseseceseseeteeceesteeeeeeeeees leaseseesseesseeaeeeseseseseaeesetenseesesenssoes 200
Figure 5.6 Microspray infusion mass spectra for peptides generated by trypsin digestion
of bovine o—casein resulting from neutral loss scanning of the Q/QITMS. No signal was
observed at m/z 644, indicating the peptide of m/z 693 was not phosphorylated. Signal was
observed at m/z 781, indicating the peptide of m/z 831 was phosphorylated.................. 206
Figure 5.7(a) Amino acid sequences corresponding to four stages of tandem mass
spectrometry of m/z 880 obtained from trypsin digestion of bovine o-casein................ 209
Figure 5.7(b) Microspray infusion mass spectra from the LCQ ion trap illustrating four
stages of tandem mass spectrometry of m/z 880 obtained from trypsin digestion of bovine
OlL-CASCIN......eeeceesscessesnececesneesceseseseseneceaesaecseeeseeseseseeseesesenetesesteeees ss eaceecassesseceeseeessnsessenensees 210
xiv
Figure 5.8 Mass spectrum and selected ion chromatogram obtained on the LCQ resulting
from the injection of 1 fmol of the peptide angiotensin I onto a positively-charged column
and eluted using capillary electrophoresis With 25 KV..........ccccccscssessessesessssessssssscescsesecsees 212
Figure 5.9 Microspray mass spectrum obtained on the LCQ resulting from loading 10
fmol of the peptide angiotensin I onto a hydrophobic membrane and eluting with 80:20:0.5
methanol: water:acetic ACid..... cece cessceseeessseeseeecssctececensacseecaeessessessssaseesensessensesseasscassasens 216
Figure 5.10 Graph of calculated signal-to-noise ratio as a function of the amount of
sample loaded onto a hydrophobic Membrane...............ccccssesssescssessseeecseseecestenscssssssessceees 218
Figure 5.11 Amino acid sequences and observed peptides from microspray analysis of a
tryptic digest Of DOVINE O-CASEIN.........eeecceecesceeceneeecssessesectessessessseseestcceeeeessaseucessenssasenses 221
Chapter 1
Introduction
1.1 Thesis Overview
The work presented in this dissertation involves the development of a number of
independent methods to analyze biological molecules by ion trap mass spectrometry.
Chapter One (below) historically presents the development of ion trap technology from a
reactor used to trap isolated ions to a versatile mass spectrometer capable of sophisticated
analysis of many different types of molecules. The particular advantages of mass
spectrometry as a means of protein sequencing are discussed and the different ionization
modes appropriate for biomolecular analysis are delineated. Strategies for protein
sequencing by ion trap mass spectrometry (1) are addressed.
Chapter Two provides a practical ‘description of the theoretical considerations
involved in ion trap operation and serves to illustrate the versatility of the instrument and
how it may be employed. Chapter Three addresses the development of a matrix-assisted
laser desorption ionization source and its use for the determination of phosphorylation sites
on the P protein of Sendai virus. A hybrid instrument composed of a quadrupole mass
filter and the quadrupole ion trap was assembled and applied to the analysis of simple
mixtures of peptides. Results are presented in Chapter Four. Membrane chromatography
techniques at high sensitivity were investigated in Chapter Five for the analysis of mixtures
of peptides using the ion trap. A summary of results and future prospects are given in
Chapter Six.
1.2 Role of Mass Spectrometry in Biological Research
Mass spectrometry is developing into an essential technique for biochemical and
biological research. The range of problems that mass spectrometry is currently being
applied to includes the analysis of post-translational modifications of proteins (2); non-
covalent protein-protein, protein-DNA, and protein-RNA interactions (3-6); study of
peptides implicated in the functioning of the immune system (7-9); and the study of
proteins involved in signal transduction pathways (10-12). The study of these processes
by mass spectrometry is significantly facilitated by information produced from genomic
sequence analysis. Model organisms are used in the study of prokaryotic and eukaryotic
cell biology, cell differentiation, and developmental biology. The information derived from
genome sequences of these organisms will aid in identifying the function, structure, and
regulation of the gene products. Identification of the functional elements involved in a
biological process will entail correlating the sequence of proteins observed in a process to
genomic sequence information contained in databases. Post-translational modifications,
especially phosphorylation, serve to regulate pathways in cell processes. Mass
spectrometry is well-suited for the identification of gene products and covalent
modifications. |
A sensitive and versatile analytical system, capable of detecting both large and
small molecules and determining aspects of molecular structure, is required to address the
complex mixtures of molecules found in these types of biological problems.
Developments over the last ten years have made the quadrupole ion trap mass spectrometer
an excellent tool for biomolecular analysis. A quadrupole ion trap is an instrument roughly
the size of a tennis ball whose size is inversely proportional to its versatility. Three
hyperbolic electrodes, consisting of a ring and two endcaps, form the core of this
instrument (Figure 1.1). Using theory to drive instrument development, the nominal mass
range of the instrument has been extended from mlz 650 to m/z 70,000 (13); up to 12
stages of tandem mass spectrometry (MS'?) have been performed (14); and mass
resolution that can allow the separation of ions of m/z 10° and m/z 10° + 1 has been
implemented (15). Quadrupole ion trap mass spectrometers are also exquisitely sensitive.
Molecular weight information has been recorded with as few as 1.5 million peptide
molecules (16). Although not all of these features can be applied simultaneously, a
judicious choice of parameters can afford sensitive molecular weight measurements and
structural analyses of biopolymers.
1.3 History of the Development of Ion Traps
In the early 1950's, Wolfgang Paul and co-workers invented two instruments that
could be used to determine mass-to-charge (m/z) ratios of ions (17, 18). The first was the
quadrupole mass filter that rapidly was applied to a wide range of analytical problems (19).
The second was the quadrupole ion trap, consisting of a ring electrode and two endcap
electrodes with hyperbolic surfaces. As is shown in Table 1.1 (20), the quadrupole ion trap
was primarily used by the physics community, notably Hans Dehmelt at the University of
Figure 1.1 Rendering of the ion trap electrode assembly showing the two endcaps and the
ring electrode.
Table 1.1 Time line of ion trap technology development.
1953
1959
1959
1962
1968
1972
1976
1978
1979
1980
1982
1983
1984
1985
1987
1990
1991
1991
1992
1993
Invention of quadrupole mass filter and quadrupole ion trap by Paul.
Storage of single microparticles.
Use as a mass spectrometer. Detection by power absorbance.
Single ions stored at low temperatures to set frequency standards.
Use as a mass spectrometer with external detection.
Characterization of the ion trap: chemical ionization, study of ion/molecule
kinetics. Used as a storage device with a quadrupole mass filter employed for
mass analysis.
Ions collisionally focused.
Used as a selective ion reactor.
Ions resonantly ejected.
Used as a GC detector.
Multiphoton dissociation of ions.
Development of mass-selective instability mode of operation.
Commercialization of ion trap detector ITD™),.
Commercialization of ion trap mass spectrometer ITMS™),
High performance mass spectrometry: multiple stages of mass spectrometry,
chemical ionization, photodissociation, external ion injection, mass range
extension.
Electrospray ionization of biopolymers.
High resolution.
Discovery of non-linear effects.
Matrix-assisted laser desorption ionization of biopolymers.
Biological problem-solving using ion trap mass spectrometry.
Washington, to investigate the properties of isolated ions (21-24). The ion trap was
operated at that time in a "mass-selective stability" mode of operation. In this mode,
analogous to the operation of a quadrupole mass filter, rf and de voltages applied to the ring
electrode were ramped to allow stability, hence storage, of a single (increasing) value of
m/z in the ion trap (20). Ions were detected by resonance absorption from an external
power source (25) or were ejected using a dc pulse applied to an endcap and detected using
an electron multiplier (26). Due to limited mass range and resolution, these methods of
mass measurement were not practical for many analytical purposes.
The chemistry community's interest in the trap was confined to several research
groups until 1983 when George Stafford and co-workers at Finnigan MAT made two
major advances. First, they developed the mass-selective instability mode of operation
(27). The fundamental difference between this mode of operation and previous methods is
that all ions created over a given time period were trapped and then sequentially ejected
from the ion trap into a conventional electron multiplier detector. Thus, all ions were stored
while mass analysis was performed, unlike the mass-selective stability mode of operation
that had been previously employed. This new method for operating the ion trap simplified
the use of the instrument. Stafford's group next discovered that a helium damping gas of ~
1 mtorr within the trapping volume greatly improved the mass resolution of the instrument
(28). Both of these discoveries led to the successful development of a commercial ion trap
mass spectrometer. In later work, the addition of helium was observed to significantly
improve trapping efficiencies, especially for externally injected ions (29). Subsequent
innovations have been rapid. Cooks and co-workers at Purdue University have pioneered
high performance techniques such as external injection of ions (29), mass range extension
(13), MS? (14), and high resolution (15) that improved the performance of the ion trap and
created interest in its application to biological molecules.
1.4 Strategies for Protein Sequencing by Tandem Mass Spectrometry
Proteins form a class of molecule that function in a key role in nearly all biological
processes. The broad range of protein interactions is due to their enormous structural
variability. Proteins are heteropolymers composed of at least 20 amino acids. The
sequential arrangement of the amino acids determines the protein structure and function;
consequently, knowledge of the amino acid sequence is the first step toward an
understanding of protein function at the molecular level.
1.4.1 Classical Microseguencing Techniques
Historically, protein sequencing has been accomplished by chemical or enzymatic
cleavage of a protein followed by separation and purification of the resulting peptides.
~ Amino-terminal sequencing of the purified peptides is performed using variations of the
method described by Edman and Begg (30) in which a series of chemical reactions are
used to cleave the N-terminal amino acid from the peptide and identify it by retention time
on a reverse phase HPLC column. The process is sequentially repeated on the shortened
peptide. Background levels and repetitive yields limit the sensitivity of this technique to the
high fmol to low pmol regime (31). The throughput of the technique is also rather slow,
with cycle times of ~ 15 min. The most significant limitation of the Edman technique,
however, is the inability to identify or sequence through post-translational modifications.
For instance, modifications such as phosphorylation are crucial regulators of signal
transduction pathways (32) and it is of great interest to identify these sites of modification.
1.4.2 Protein Sequencing By Tandem Mass Spectrometry
Since proteins are heteropolymers of distinct masses (Table 1.2), mass
spectrometry is a logical tool for primary structure analysis. Molecules of biological origin
are typically highly polar and thermally labile, thus were not compatible with conventional
ionization techniques such as electron impact (EI) and chemical ionization (CI). Three new
ionization techniques developed in the 1980's were employed to effectively ionize
biological molecules and, when interfaced with mass spectrometry, revolutionized the
analysis of biomolecules.
1.4.3 Ionization Techniques for Biomolecules
1.4.3.1 Ionization By Atom/Ion Bombardment
In fast atom or ion bombardment (33), termed liquid secondary ion mass
spectrometry (LSIMS), the sample is dissolved in a viscous liquid matrix such as
monothioglycerol then bombarded with a 6-8 keV beam of atoms or ions. Peptide ions
residing on the surface of the matrix are sputtered into the gas phase. Diffusion of
molecules from the bulk solution serves to replenish the surface and ensure a steady
current of sample ions. LSIMS mass spectra are characterized by the presence of an
abundant (M+H)* ion and a low abundance of fragment ions. The spectra also contain a —
g P
10
Table 1.2 Abbreviations and incremental masses for the 20 commonly occurring amino
acids. Mass is included for phosphorylation of serine.
Amino Acid and One Letter Incremental
Abbreviation Code Mass*
Glycine - Gly G 57.05 ©
Alanine - Ala A 71.08
Serine - Ser S 87.08,
Phosphoserine - Psr S 167.06
Proline - Pro P 97.11
Valine - Val Vv 99.13
Threonine - Thr T 101.10
Cystine - Cys C 103.14
Leucine - Leu L 113.16
Isoleucine - Ile I 113.16
Asparagine - Asn N 114.10
Aspartic Acid - Asp D 115.09
Glutamine-Gin Q 128.13
Lysine - Lys K 128.17
Glutamic Acid - Glu E 129.11
Methionine - Met M 131.19
Histidine - His H 137.14
Phenylalanine - Phe F 147.18
Arginine - Arg R 156.18
Tyrosine - Tyr Y 163.18
Tryptophan - Trp WwW 186.21
“corresponds to the formula -NHCHRCO- where R indicates the side chain characteristic of the
particular amino acid.
11
relatively high background of ions derived from the matrix and sample ions that suffer
radiation damage from prolonged exposure to the projectile beam (1). The technique is
useful for analyzing a collection of peptides present in mixtures and has been used to
characterize the molecular weights of peptides generated in tryptic mapping experiments.
A limitation of the method is that signal from hydrophilic peptides is much less abundant
than signal from hydrophobic peptides due to the presence of an excess of the latter on the
surface of the matrix.
1.4.3.2 Matrix-Assisted Laser Desorption Ionization
In matrix-assisted laser desorption ionization (MALDI) (34, 35), the sample is
dissolved in an excess (~10%:1 of matrix:sample) of an acidic liquid matrix and air dried.
The matrix and sample co-crystallize. Some common matrices used are 2,5-
dihydroxybenzoic acid, -cyano-4-hydroxycinnamic acid, or sinapinic acid (36-39).
Typically, laser light from the frequency quadrupled ouput of a Nd:YAG laser at 266 nm
or the output of a nitrogen laser at 337 nm is directed onto the crystals. The benzene ring
of the matrix strongly absorbs the photons and desorbs into the gas phase, carrying the
sample off the probe. Proton transfer is thought to occur within the ion plume, although
the mechanism for this is not well understood. Ionized matrix and sample are directed into
the mass spectrometer. MALDI mass spectra are characterized by the presence of an
abundant (M+H)* ion and a low abundance of fragment ions depending upon the matrix
used. The spectra also contain a relatively high background of ions derived from the
matrix. The technique is useful for analyzing a collection of peptides present in mixtures
and has been used to characterize the molecular weights of peptides generated in tryptic
12
mapping experiments. Signal suppression is more limited than that encountered when
utilizing LSIMS techniques; thus MALDI is in more general use as a method for analyzing
mixtures of peptides. In addition, MALDI is capable of ionizing very large proteins (~1
MDa) (40, 41) and has been employed for the analysis of oligonucleotides (42) and
proteins (43-46) as well as for the analysis of peptide mixtures.
1.4.3.3 Electrospray Ionization
In electrospray ionization (47, 48), the sample is diluted in an acidic liquid matrix,
typically 0.5% acetic acid or 0.1% trifluoroacetic acid. Methanol is added to the mixture
and the resultant solution is directed into a needle. The needle is placed at a high potential
(1-5 kV) and is brought close to a capillary (often heated) that is held near ground. Strong
electrical fields near the tip of the needle induce droplet formation by electrohydrodynamic
shearing of the charged liquid. The droplets begin to desolvate. When the force due to the
surface charge density is sufficient to overcome the surface tension forces, the droplets
"explode" into a number of smaller droplets. At the final stage, ions desorb from the
droplets into the gas phase (49, 50). Electrospray ionization mass spectra typically contain
a distribution of multiply-charged species. Often the singly-charged ion is not observed,
thus MS/MS of peptides is typically accomplished on the doubly- or triply-charged ion.
Signals from matrix are very low in abundance; thus the mass spectra have improved
signal-to-noise ratios when compared with LSIMS and MALDI. The primary advantage
of the ESI technique is that it can be easily interfaced to conventional peptide separation
devices such as reverse-phase high performance liquid chromatography (HPLC) (51) or
capillary electrophoresis (CE) (52, 53) that afford the separation of peptide mixtures.
13,
Separation with subsequent analysis by mass spectrometry can be performed "on-line,"
reducing sample handling losses.
1.4.4 Sample Preparation
Proteins are subjected to chemical or enzymatic digestion to produce a mixture of
peptides. The mixture is then fractionated by HPLC. This separation technique resolves
peptides according to their hydrophobicity as well as their size. If LSIMS or MALDI are
employed as the ionization technique, the presence of peptides in the eluent from the HPLC
is monitored by ultraviolet (UV) absorbance and fractions corresponding to peaks in the
UV chromatogram are collected for further analysis. If ESI is employed, the eluent is
directed through a fused silica capillary into the electrospray needle and the separated
peptide mixture is subsequently mass analyzed.
1.4.5, Fragmentation of Peptides in Low Energy CID Processes
To determine the amino acid sequence of a peptide, the molecular weight is
recorded, then a fragmentation mass spectrum is collected. Peptide ions are trapped then a
population of ions with a given m/z value is selected and resonantly excited. The ions
undergo tens of thousands of collisions with neutral helium atoms within the trapping
volume and become vibrationally excited. Fragmentation subsequently occurs primarily at
the amide bonds, producing a ladder of sequence ions (1). The resulting charged
fragments are mass analyzed to produce a mass spectrum characteristic of the original
peptide structure. Knowledge of the fragmentation patterns of peptides under low energy
14
collision conditions allows the amino acid sequence to be reconstructed from the
fragmentation mass spectrum (1).
Four major types of fragment ions are produced in the collision process. A
summary of these pathways is shown in Scheme I (1). If the amount of kinetic energy
converted to vibrational energy is high, cleavage of a single bond typically results and
acylium ions of type "b" are produced (Pathway (1)). If the energy transferred is low,
simultaneous bond formation and bond cleavage is favored and ions of type "y" result
(Pathway (2)). Cleavage of at least two bonds internal to the peptide chain can also occur
giving rise to ions with the general formula NH,=CHR* or NH,CHRCO* and are
designated as type "a" (Pathway (3)). Ions of this type often dominate the low mass end of
the spectrum. Not all amino acid residues in a peptide afford this type of fragment ion.
Finally, multipoint cleavage of the peptide can produce ions with the same formula as type
b ions, shown in Pathway (4). These ions are designated as “b,y,," where b, and y,,
indicate the points of cleavage to produce the carboxyl and amino termini of the fragment.
Proline-containing peptides generate ions of this type with a higher frequency than other
amino acid residues (1). Internal cleavage products can also be generated by arginine and
histidine containing peptides. The major fragment ions ‘likely to be produced in the
collision activation process can be predicted for a known amino acid sequence.
1.4.6 Data Interpretation
Interpretation of peptide collision activated dissociation mass spectra is based on the
above mechanistic considerations and is outlined in detail in a review by Yates et al. (54).
When electrospray ionization is employed, peptides are generally created by trypsin
15
Scheme 1: Dominant collision-induced fragmentation pathways for peptides.
‘16
Rr ; Ra . R
ae — ‘n,7 * co wey by,
8) (4) A
NH NH NH NH. ~~ ~NH~ 0H
on ae: r :
Re 4
Ry oH Ra HH
R Oo. UR
+? +
-O Rs oO Rs
NH Ny oH Ry NH OH Y2
Ry 0
Ry 6) 5
b, MeN + NHs OH y,
Am/z = mi AOm/z = Nay
Scheme I
17
proteolysis and form doubly-charged ions. This stems from the presence of basic sites at
both the N- and C-terminus in the form of the &-amino group and the basic amino acids
Lys or Arg. Most of the CID-generated fragment ions are singly-charged. In general, a
ladder of sequence ions is produced and the mass of an amino acid present at a given
position in the sequence can be deduced from subtracting the masses of consecutive ion
signals. Successful interpretation involves determining which ions are of type y and which
are of type b so that mass differences between consecutive ions of the same type can be
calculated. The masses of the largest amino acid, Trp (186 u), and the smallest amino acid,
Gly (57 wu), are used to create a mass window with which to interrogate the mass spectrum.
Given a doubly-charged precursor ion, the mass of the singly-charged analog can be
calculated. The largest ion of type y is found within the mass window below the precursor.
The largest ion of type b is found within the mass window beginning at 18 u below the m/z
of the precursor. If a signal corresponding to an ion of type b is generated, the spectrum is
searched for the presence of the smallest ion of type y in the low mass region of the
spectrum. If the peptide results from trypsin digestion, this ion will be Lys or Arg with
m/z 147 or 175. These low mass ions are typically not observed in an ion trap mass
spectrometer for reasons discussed in Chapter Two. The process is continued. The mass
window is employed to interrogate the spectrum in the region below the identified peak, a
new peak is identified, and confirmation is sought in the form of the presence of the
complementary ion. Often a number of possibilities exist and must be continued until no
candidate sequence ions can be determined.
Several methods can be used to verify the deduced sequence (54). Derivitizing the
peptide by creating the methyl ester will add methyl groups to all of the acidic residues, as
18
well as the C-terminus, thus the mass of the peptide should shift and the masses of all of
the ions assigned as type y should shift by 14 u. Additional 14 u shifts in mass indicate the
presence of Glu, Asp, or S-carboxymethyl Cys. Acetylation of an unblocked N-terminus
shifts the mass of the peptide and that of the b-type ions by 42 u. Any additional 42 u
increments indicate the presence of Lys in the peptide. Losses of CO from b ions and
losses of water from peptides containing Ser or Thr may also add confidence to sequence
assignments.
1.4.7 Analysis of MS Data Using Known Sequences
Information generated by mass spectrometry can be correlated to sequences in
genome databases to aid in protein identification or mass spectral interpretation as
discussed in a recent review (54). This information may be useful for screening new data
to determine if it is already contained in the sequence database and for investigation of
known proteins utilizing different experimental contexts.
1.4.7.1 Peptide Mass Mapping
By digesting a protein with site-specific enzymes, the calculated masses of the
_ predicted peptide products from the gene sequence can be compared with those observed
experimentally (54). This method is useful for identifying the presence of post-
translational modifications and sequence or translation errors. If the protein being studied
is not known, the molecular weights of the enzymatically-generated fragments can be
compared with calculated fragments from other protein sequences in the database under the
19
same digestion conditions. The unknown protein can be identified with high probability if
the peptide maps match. A number of computer programs can be employed to perform
mass map database searches (2, 54). Mass tolerances as large as 5 u and as small as 7% of
the total protein mass have been used to identify proteins (54). The sensitivity of the
technique is such that proteins may be correctly identified from ‘highly similar protein
families. Proteins with highly similar sequences may produce very diverse peptide maps
(54). Post-translational modifications will only change the mass of the modified peptide
and will not affect the rest of the mass map. The technique has proven useful for
identifying proteins isolated from two-dimensional gel electrophoresis (55).
1.4.7.2. Computer-Aided Interpretation of Fragmentation Mass Spectra
A peptide mass map produces a fingerprint by which proteins can be identified.
Similarly, a fragmentation mass spectrum produces a fingerprint by which a peptide
sequence can be identified (54).. Manual interpretation of CID mass spectra is tedious and
provides a bottleneck in the ultimate throughput of the mass spectrometric analytical
technique. Fortunately, a number of algorithms have been developed to afford computer-
aided interpretation of CID mass spectra (2, 54). SEQUEST, an algorithm developed by
Yates and Eng (56), is used in portions of this dissertation for peptide identification. The
program takes the molecular weight of the peptide and searches a database for all character-
based sequences of amino acids whose molecular weights add up to that of the observed
peptide, within a small error tolerance. Expected b- and y-type ions are calculated for each
sequence and compared with the dominant peaks in the experimental mass spectrum,
generating a preliminary score. For the top 500 candidates, a theoretical mass spectrum is
20
generated then cross-correlated with the experimental mass spectrum and ranked. The
answers generated are compared manually with the experimental mass spectrum, and the
validity of the sequence assignment is determined. A search can be carried out on all
sequences or just those defined by the proteolytic specificity of an enzyme or a partial
amino acid sequence. Data analysis is rapid and completely automated using this software
and is fast. Searching a database containing 100,000 sequences takes 2-3 min on a
DecStation 3000/9000 computer. The algorithm also includes the capability for searching
for sequences with user-defined post-translational modifications (57, 58).
1.4.7.3 de novo Computer Interpretation
Tandem mass spectrometers, especially when coupled to LC, have an enormous
throughput and generate huge amounts of data. If a given fragmentation spectrum does not
appear to correlate well with a peptide sequence in the database, the spectrum can be
interpreted manually or by a de novo computer interpretation algorithm. A combinatorial
approach is usually used (54). Starting at the C-terminus, amino acid masses are
subtracted from the (M+H)* ion to calculate the y- and b-type ions that would be present
for each amino acid at that position in the sequence. A score is calculated based on the
abundance of ion signal for each possibility, and the 20 amino acids are ranked. The
second iteration creates a list of 400 possibilities by extending each amino acid by an
additional one. This list is ranked and only sequences with a non-zero score will be
retained in the next iteration. The process is continued until the calculated sequence mass
matches the m/z for the observed (M+H)* ion. This approach has been used to interpret
high quality data (54).
21
_1.5 Conclusion
Mass spectrometry can be used to obtain information about biological molecules,
hence biological processes, with a faster throughput and at higher sensitivities than can be
accomplished using conventional methods such as Edman degradation. The work
presented in this dissertation involves the development of methodologies to extend the
capabilities of the versatile ion trap mass spectrometer for the analysis of peptide mixtures
and post-translational modifications.
22
1.6 References
1. Yates, J. R., HI (1987), Ph.D. Dissertation, University of Virginia, and the
references therein.
2. Burlingame, A. L., Boyd, R. K., and Gaskell, S. J. (1996) Anal. Chem. 68, 12,
599R-651R, and the references therein.
3. Smith, R. D., and Light-Wahl, K. J. (1993) Biol. Mass Spec. 22 493-501.
4. Smith, D. L., and Zhang, Z. Q. (1994) Mass Spec. Rev. 13, 5-6, 411-429.
5. Smith, R. D., Cheng, X. S., Chen, R., and Hofstadler, S. A. (1996) ACS Symp.
Ser. 619 294-314. |
6. Przybylski, M., and Glocker, M. O. (1996) Angew. Chem. Int. Ed. Engl. 35 806-
826.
7. Henderson, R. A., Michel, H., Sakaguchi, K., Shabanowitz, J., Appella, E., Hunt,
D. F., and Engelhard, V. H. (1992) Science 255 1264-1266.
8. Cox, A. L., Skipper, J., Chen, Y., Henerson, R. A., Darrow, T. L., Shabanowitz,
J., Engelhard, V. H., Hunt, D. F., and Slingluff, C. L., Jr. (1994) Science 264 716-719.
9. Slingluff, C. L., Jr., Hunt, D. F., and Engelhard, V. H. (1994) Curr. Op. Immun.
6 733-740.
10. Watts, J. D., Affolter, M., Krebs, D. L., Wang, R. L., Samelson, L. E., and
Aebersold, R. (1994) J. Biol. Chem. 269, 47, 29520-29529.
23
11. Watts, J. D., Affolter, M., Krebs, D. L., Wange, R. L., Samelson, L. E., and
Aebersold, R. (1996) ACS Symp. Ser. 619 381-407.
12. Affolter, R., Watts, J. D., Krebs, D. L., and Aebersold, R. (1994) Anal. Biochem.
223 74-81.
13. Kaiser, R. E., Jr., Cooks, R. G., Stafford, G. C., Jr, Syka, J. E. P., and
Hemberger, P. H. (1991) Int. J. Mass Spectrom. Ion Proc. 106 79-115.
14. Louris, J. N., Brodbelt-Lustig, J. S., Cooks, R. G., Glish, G. L., Van Berkel, G. J.,
and McLuckey, S. A. (1990) Int. J. Mass Spectrom. Ion Proc. 96 117-137.
15. | Cooks, R. G., Hoke, S. H., I, Morand, K. L., and Lammert, S. A. (1992) Int. J.
Mass Spectrom. Ion Proc. 118/119 1-36.
16. Kaiser, R. E., Jr., Cooks, R. G., Syka, J. E. P., and Stafford, G. C., Jr. (1990)
Rapid Commun. Mass Spectrom. 4, 1, 30-33.
17. Paul, W., and Steinwedel, H. (1956) ,. German Patent 944,900.
18. Paul, W. (1990) Angew. Chem. Int. Ed. Engl. 29 739-748.
19. Cooks, R. G., McLuckey, S. A., and Kaiser, R. E. (1991) Chem. Eng. News 69,
12, 26-41.
20. March, R. E., and Hughes, R. J. (Eds.) (1989) Quadrupole Storage Mass
Spectrometry, (Winefordner, J.D. and Kolthoff, I.M., Eds.) in Chemical Analysis: A
Series of Monographs on Analytical Chemistry and its Applications, V102, Wiley &
Sons, New. York.
24
21. Dehmelt, H. G. (1967) Adv. At. Mol. Phys. 3 53-72.
22. Dehmelt, H. G. (1969) Adv. At. Mol. Phys. 5 109.
23. Dehmelt, H. (1991) in Santa Fe Workshop. Foundations of Quantum Mechanics
(Black, T. D., Nieto, M. M., Pilloff, H. S., Scully, M. O., and Sinclair, R. M., Eds.), May
27-31, 1991, World Scientific, Singapore, Santa Fe, NM, USA, pp. 16-22.
24. Dehmelt, H. (1995) Phys. Scripta Vol. T T59 87-92.
25. Rettinghaus, V. von G. (1967) Z. Angew. Phys. 22, 4, 321-326.
26. Dawson, P. H., and Whetten, N. R. (1968) J. Vac. Sci. Technol. 5,1, 11-8.
27. Stafford, G. C., Jr., Kelley, P. E., Syka, J. E. P., Reynolds, W. E., and Todd, J. F.
J. (1984) Int. J. Mass Spectrom. Ion Proc. 60 85-98.
28. Louris, J. N., Cooks, R. G., Syka, J. E. P., Kelley, P. E., Stafford, G. C., Jr., and
Todd, J. F. J. (1987) Anal. Chem. 59 1677-1685.
29. Louris, J. N., Amy, J. W., Ridley, T. Y., and Cooks, R. G. (1989) Int. J. Mass
Spectrom. Ion Proc. 88 97-111.
30. Edman, P., and Begg, G. (1967) Eur. J. Biochem. 1 80-91.
31. Hewick, R. M., Hunkapiller, M. W., Hood, L. E., and Dreyer, W. J. (1981) J.
Biol. Chem. 256 7990-7997.
32. ‘Hunter, T. (1995) Cell 80 225-236.
33. Barber, M., Bordoli, R. S., Sedgwick, R. D., and Tyler, A. N. (1981) J. Chem.
Soc. Chem. Commun. 325-327.
25
34. Karas, M., Bachmann, D., Bahr, U., and Hillenkamp, F. (1987) Int. J. Mass
Spectrom. Ion Proc. 78 53-68.
35. Tanaka, K., Waki, H., Ido, Y., Akita, S., and Yoshida, Y. (1988) Rapid Commun.
Mass Spectrom. 2 151-153.
36. Schar, M., Bornsen, K. O., and Gassman, E. (1991) Rapid Commun. Mass
Spectrom. 5 319-326.
37. Beavis, R. C., and Chait, B. T. (1990) Proc. Natl. Acad. Sci. USA 87 6873-6877.
38. Cohen, S. L., and Chait, B. T. (1996) Anal. Chem. 68 31-37.
39, Strupat, K., Karas, M., and Hillenkamp, F. (1991) Int. J. Mass Spectrom. Ton
Proc. 111 89-102.
40. Nelson, R. W., Dogruel, D., and Williams, P. (1994) Rapid Commun. Mass
Spectrom. 8 627-631.
41. Nelson, R. W., Dogruel, D., and Williams, P. (1995) Rapid Commun. Mass
Spectrom. 9 625.
42. Limbach, P. A., Crain, P. F., and McCloskey, J. A. (1995) Curr. Opin.
Biotechnol., 6 96-102.
43. Colby, S. M., King, T. B., and Reilly, J. P. (1994) Rapid Commun. Mass
Spectrom. 8 865-868.
44. Whittal, R. M., and Li, L. (1995) Anal. Chem. 67 1950-1954.
45. Brown, R. S., and Lennon, J. J. (1995) Anal. Chem. 67 1998-2003.
26
46. Vestal, M. L., Juhasz, P., and Martin, S. A. (1995) Rapid Commun. Mass
Spectrom. 9 4144-1050.
47. Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F., and Whitehouse, C. M. (1989)
Science 246 64-71.
48. Fenn, J. B., Mann, M., Meng, C. K., and Wong, S. F. (1990) Mass Spec. Rev. 9
37-70.
49. Iribarne, J. V., and Thomson, B. A. (1976) J. Chem. Phys. 64 2287-2294.
50. Fenn, J. B. (1993) J. Am. Soc. Mass Spectrom. 4524-535.
51. Whitehouse, C. M., Dreyer, R. N., Yamashita, M., and Fenn, J. B. (1985) Anal.
Chem. 57 675-679.
52. Lee, E. D., Mueck, W., Henion, J. D., and Covey, T. R. (1988) J. Chromatogr.
458 313-321.
53. Smith, R. D., Loo, J. A., Barinaga, C. J., Edmonds, C. G., and Udseth, H. R.
(1989) J. Chromatogr. 480 211-232.
54. Yates, J. R., I, McCormack, A. L., Link, A. J., Schieltz, D., Eng, J., and Hays, L.
(1996) Analyst 121, 7, R65-R76, and the references therein.
55. Henzel, W., Billeci, T., Stults, J., Wond, S., Grimley, C., and Watanabe, C. (1993)
Proc. Natl. Acad. Sci. USA 90 5011-5015.
56. Eng, J., McCormack, A. L., and Yates, J. R., TI (1994) J. Am. Soc. Mass
Spectrom. 5 976-989.
27
57. Griffin, P. R., MacCoss, M. J., Eng, J. K., Blevins, R. A., Aaronson, J. S., and
Yates, J.R., If (1995) Rapid Commun. Mass Spectrom. 9 1546-1551.
58. Yates, J. R., III, Eng, J., McCormack, A. L., and Schieltz, D. (1995) Anal. Chem.
67 1426-1436.
28
Chapter 2
Practical Aspects of Ion Trap Theory
2.1 Theoretical Overview
Quadrupole ion traps are dynamic mass analyzers that use an oscillating electric
potential applied to the ring electrode, called the “fundamental rf,” to focus ions toward the
center of the trap. This is accomplished by creating a parabolic potential, shaped like a
saddle (1), inside the trapping volume. The strength of the restoring force linearly
increases as the ion trajectory deviates from the central axis, focusing the ion back to the .
center of the trapping volume. This is demonstrated in Figure 2.1, a simulation of ion
trajectories created using SIMION 3D Version 6.0 (2). A population of trapped ions is
observed to occupy only the space near the center of the trap due to the focusing effect of
the oscillating electric fields. Assuming a cylindrically symmetric system, the potential an
ion experiences at any point in the ion trap is given by
D(r,z) =
Baoan e =e. (U — Vos ar) (Eq. 2.1)
2 r 2
where U is the amplitude of a dc potential applied to the endcap electrodes with reference to
the ring electrode, Vis the amplitude of the "fundamental rf’ applied to the ring electrode,
® is the angular frequency of the rf potential and r, is the closest distance between the
29
Figure 2.1: Simulation of ion trajectories in the ion trap using SIMION. 3D. The ion
trajectories quickly collapse toward the center of the trap.
30
31
center of the trap and the ring electrode (3). The closest distance between the center of the
trap and the endcap electrode is given by Z,. To obtain an ideal quadrupolar field, r, is
equal to the square root of 2z,. The actual geometry of the commercial ITMS is
"stretched" and r, is equal to 0.781Z,, leading to the presence of higher-order fields within
the trapping volume. The effects of non-linear resonances produced in the stretched trap
have been actively studied for the last few years and have led to many new insights
regarding the fundamental performance characteristics of the ion trap mass spectrometer
(4-10).
The force on an ion, given by the electric field, is obtained by
F(r,z) = E(r,z) =—eV®(r,z) = malr,z) (Eq. 2.2)
and, using Newton's law, is proportional to the acceleration an ion of charge e€ experiences
due to that force. Equation 2 may be placed in the form of the Mathieu equation (11) in the
radial and axial directions when the substitutions
—8eU | 4eV a q ot |
= ; =——, =o £4 > f =s— 4Z > d =e E ° 2.3
a, mr? q. mr a 2° 1 VA “ 6 2 (Eq )
are made. Ion trajectories are determined by solutions to the Mathieu equation and are
oscillating functions with regions of stability described by the parameters a, and g,. Thus |
the stability of ion motion depends upon the mass and charge of the ion (m), the size of
32
the ion trap (7,), the oscillating frequency of the fundamental rf (@), and the amplitudes of
the applied de (U) and rf (V) voltages. One region of stability in which radial and axial
stability overlap is shown in Figure 2.2. An ion of a given mass-to-charge ratio will be
stably trapped anywhere within that region. The position of the ion within the stability
region can be moved by changing the amplitude of the applied dc and rf voltages to change
the values of a, and q_, termed the “working points” of the ion. Values of the working
points are chosen to ensure stability or instability of an ion trajectory of interest. For the
case of the commercial Finnigan ion traps, 7,= 1 cm, @/2n = 1.1 MHz, with V ranging
from 0 - 7500 V,,,.
- As an example, consider three working points for an ion of m/z 1500, shown in
Figure 2.3. Values of the amplitudes for the applied dc and rf potentials are shown in
parentheses. The corresponding @, and q, values are delineated in the figure caption. It is
clear that a judicious choice for the amplitude of the applied potentials is required to ensure
stability for all ions within a mass range of interest. The mass-selective instability mode of
operation utilizes no dc voltage, thus the mass spectrometer is operated on the line a, = 0.
This corresponds to the case of maximizing the range of m/z values that may be stably
trapped. Jon trajectories become unstable in the axial direction (between the endcap
electrodes) but remain stable in the radial direction when g, = 0.908. Ions are ejected
through holes in the endcap electrode and are typically detected using an electron multiplier.
Trapped ions of a given m/Z oscillate at a frequency known as the “secular
frequency” that is proportional to the angular frequency of the applied signal, m. The
33
Figure 2.2: Diagram showing the regions of stability in the quadrupole ion trap
parameterized in terms of the operating voltages and frequencies.
34
35
Figure 2.3: Selected "working points" for an ion of m/z 1500. The applied de and rf
potentials are shown in parentheses, (U, V). The corresponding (@,, g, ) values are as
follows: (-100 V, 1000 V) = (0.0108, 0.0539), similarly (-1000 V, 3000 V) => (0.108,
0.162) and (-100 V, 6000 V) = (0.0108, 0.323). A judicious choice of conditions is
required to ensure trajectory stability for a wide range of m/z values.
0.2
- 0.2
36
@ (1K3K)
(-0.1 K,1 K)
qz
37
constant of proportionality is given by B,,. For values of g, < 0.4, B, may be
- approximated by (12),
, 2
Baa,+%h (Eq. 2.4)
which reduces to B, = 1/5 for the mass-selective instability mode of operation.
“Resonance” conditions are induced by matching the frequency of a supplementary
potential applied to the endcap electrodes to the secular frequency of the ion. The ion will
absorb energy from the applied field and the trajectory will linearly increase towards the
endcap electrodes until the ion becomes unstable and is ejected (3).
2.2 Practical Aspects of Ion Trap Theory
In order to measure the m/z value of a molecule in an ion trap, the molecule must —
be ionized, focused into the ion trap, trapped, ejected, and detected. Structural information
is obtained by collision-induced dissociation with a helium damping gas, and a mass
spectrum is generated by sequentially ejecting fragment ions from low m/z to high m/z.
The mass-selective instability mode is utilized for ion ejection. The mass-selective
instability line is the locus of g, values where @, is set to zero and maximizes the mass
range that may be stably trapped. Operation of the ion trap consists of the construction of a
scan function used to manipulate the working points of ions of interest. The scan function
sets the amplitude of the fundamental and supplementary potentials and sets the time taken
for each step. Typical scan functions for molecular weight analyses and MS/MS
experiments are shown in Figure 2.4.
38
Figure 2.4: (a) Molecular weight and (b) MS/MS scan functions for the quadrupole ion
trap mass spectrometer.
39
MS Scan
amplitude
A - Ionization Period
B - Cooling Time
C - Resonance Ejection Ramp
MS/MS Scan
if
amplitude
A - Ionization Period
B - Reverse Scan to eject high mass ions
C - Forward Scan to eject low mass ions
D - Resonance Excitation (Tickle) Period
E - Cooling Time
F - Resonance Ejection Ramp
40
2.2.1 lon Injection
fon traps were initially utilized to analyze volatile samples by electron impact or
chemical ionization. In this case, ions were created inside the trapping volume. An interest
in the analysis of biological molecules led to the need to interface suitable ionization
techniques, i.e., electrospray ionization and matrix-assisted laser desorption ionization, to
the ion trap. These externally created ions need to be injected into the ion trap and
efficiently trapped. Ions are focused by an einzel lens system and allowed into the ion trap
during the ionization period. A gating lens pulses from positive to negative voltages to
repel or attract ions toward the entrance endcap aperture. The time during which ions are
allowed into the trap is set to maximize signal while minimizing "space-charge" effects,
resulting from too many ions in the trap, that lead to an overall reduction in performance.
The ion trap is typically filled with helium to a pressure of ~ 1 mtorr. Collisions with
helium reduce the kinetic energy of the ions and serve to quickly contract trajectories
toward the center of the ion trap, enabling trapping of injected ions. This cooling effect is
demonstrated in Figure 2.5 where the ion population forms a “packet” near the center of
the trap.
2.2.2_lon Trapping
Ions of different m/z values may have stable orbits at the same time, as shown in
Figure 2.6. From the expression for g, in Equation 2.3, we see that
Vv
te. (Eq. 2.5)
z q,
41
Figure 2.5: Simulations show that collisions with the helium damping gas lead to the
creation of an ion packet near the center of the trap.
42
43
Larger values of m/z will have smaller values of g, and smaller values of m/z will have
larger q, values. Since ion trajectories become unstable when q, = 0.908, a well-defined
low-mass cutoff is created for a given value of the amplitude of the applied rf voltage, V.
No ions below that mass will be trapped, but ions above that mass will be trapped with
trapping efficiency decreasing for larger m/z values (1). Low-mass cutoffs for various
amplitudes of the applied fundamental rf potential are listed in Figure 2.6. The trapping
efficiency for an ion of interest depends, in part, upon the value of the low-mass cutoff, or
the so-called exclusion limit (6). This can be a problem when using ionization methods
that generate many low-mass matrix ions since the ion trap can accommodate on the order
of 10° ions before space-charge seriously impairs the performance of the instrument. For
example, the model peptide human angiotensin I (MW 1296) may be most efficiently
trapped at a low-mass cutoff of 85 u. Matrix-assisted laser desorption ionization (MALDD
generates matrix ions above this cutoff in a ratio of ~1:10°. In this situation, the high
sensitivity of the ion trap can be most effectively utilized if the ion of interest is selectively
injected into the ion trap. Current efforts revolve around selective injection utilizing shaped
excitation waveforms (13) or filtered noise fields (14) to cause all ions but the ion of
interest to have unstable trajectories. Other approaches include ramping the amplitude of
the fundamental rf during injection to increase trapping efficiency, even at low pressures of
the helium damping gas (15), as well as the addition of a quadrupole mass filter to afford
selective injection of ions of interest into the ion trap (16).
44
2.2.3 lon Ejection
Shown in Figure 2.6 is an example of the relative positions of three ions of
differing m/z ratios on the mass-selective instability line, a,= 0. Three different values for
the amplitude of the fundamental rf signal are given. As the voltage is increased, the q,
value for the ion also increases. Figure 2.6(c) shows that at 6000 V, the ion of m/z 500 has
been ejected from the ion trap. At the maximum amplitude of 7500 V, at m/z 1500 the q,
value has only reached 0.404; thus that ion cannot be ejected from the ion trap and detected.
As noted above, a resonance condition may be induced by matching the frequency of an
applied oscillating signal to the secular frequency of an ion in the trap (17). This will cause
an ion to gain energy and the amplitude of the trajectory to linearly approach the endcap
electrodes until the ion is ejected from the trap. Ejection can therefore be made to occur at
voltages lower than those required for ejection at g, of 0.908, extending the nominal mass
range of the ion trap. Conceptually, this may be viewed as creating a “hole” in the stability
diagram. The position of the hole is dependent upon the frequency of the supplementary
potential while the size of the hole depends upon the amplitude of the signal. This effect is
illustrated in Figure 2.7 where an ellipse represents a resonance point that extends the mass
range by a factor of 4. At 1000 V none of the ions have g, values approaching that of the
resonance point; thus none will be detected. At 3000 V, m/z 500 has been ejected and m/z
1000 is in the process of being ejected. The q, value for m/z 1500 is smaller than 0.227;
thus that ion will not be ejected. At 6000 V, the g, values for all of the ions are greater than
0.227, the g, value of the resonance point. This example shows that when resonance
45
Figure 2.6: Relative positions of ions with three different mass-to-charge ratios along the
mass-selective instability line, a, = 0. The effect of increasing the amplitude of the
fundamental rf voltage is shown in panels (a) through (c).
46
> 3060 LI8O 9cL0 9€90 SSO SPO. £90 7270 7810 1600
N PES WU] WoIsN]oxa
A 0009=A
8060 LI8O 9ZL'0 9£9°0 Svs0 vsv0 £9t'0 cLE0 c8T0 160°0
N £97 HUW] WOIsNpoxa
A 000€=A
2060 LIs0 9cL0 9£90 SPO PSVO f9€0 CLTO C810 1600
N 6§ JIU] UOISNDx9
00ST zu @
0001 zu @
00S Z/U ee
A 0001=A
(9
(eV
47
Figure 2.7: The same conditions as in Figure 2.6 except a resonance point at g, = 0.227
has been imposed to increase the effective mass range by a factor of 4. A region of
instability is created that affords the ejection of ions at lower voltages than would normally
be required; therefore, ions of large m/z can be ejected from the ion trap and detected.
48
*b 8060 LI80 9¢L0 990 ShSO PSO £9E0 TZLZ0 780. 1600
~«t tC
A 0009=A
2b 806'°0 LIS'0 97L0 9F90 SSO PSrO €9€0 CLZO Z8I0 T[é60'0
~_ | | | | | | | i ae
A 000€=A
2b 8060 LI80O 9CL0 9£9°0 Eco. rSvO €9€0 ZLZ0 7810 160°
~ +—C>+ \
00ST zu @
OOOT Zur © A. 000T=A
00S Z/ur e
49
ejection is used and the amplitude of the voltage is ramped from low to high amplitudes, all
of the ions “fall through the hole” and are ejected from the trap and detected.
2.2.4 Ion Isolation
In a typical multiple-stage mass spectrometry experiment, the ion of interest is
isolated before undergoing resonance excitation or charge state determination using high
resolution. Isolation in the Finnigan ITMS may be accomplished in two ways, depicted in
Figure 2.8. One method, illustrated in Fig. 2.8(a), includes the combined use of dc and rf
potentials to bring the q, and @, values of the ion to an apex of the stability diagram; all
other ions will be unstable (18, 19). The other method is shown in Fig. 2.8(b) and
consists of scanning the amplitude of the fundamental rf voltage in a reverse-then-forward
manner while applying a resonance signal (20, 21). This allows ejection of ions with m/z
greater than the ion of interest followed by ejection of ions having m/z smaller than the ion
of interest. Both isolation methods are used; however, the effects of space-charge and field
non-linearities on the shape of the stability diagram may degrade performance when the
dc/rf isolation method is employed. A recent refinement includes the use of the stored
waveform inverse Fourier transform (SWIFT) technique (13, 22) and filtered noise fields
(14) to isolate ions using notched waveforms.
2.2.5 lon Dissociation
As discussed above, when an ion approaches a region of instability in the axial
direction, the deviation of its trajectory from the center of the trap will increase. When
50
Figure 2.8: Methods of isolating a single m/z in an ion trap. (a) A combination of de and
rf potentials are applied to bring the a, and q, values of the ion of interest to the apex of the
stability diagram. Neighboring ions have working points that fall outside of the region of
stability. (b) Reverse-then-forward scanning of the amplitude of the fundamental rf
voltage in conjunction with the application of an auxiliary signal to create a resonance point
affords ion isolation. (i) Reverse scanning resonantly ejects ions from high to low m/z. —
' (ii) Forward scanning resonantly ejects ions from low to high m/z. (iii) Resultant
isolation of one value of m/z.
51
52
instability is induced by a resonance signal, the amplitude of the resonance signal can be
adjusted to cause collisionally-induced dissociation (CID) of the ion with the helium
damping gas rather than ejection from the ion trap (20). An estimated 10,000 low-energy
collisions (23) transfer enough energy into peptide ions to cause random fragmentation
along the peptide backbone in a manner analogous to that obtained using a triple
quadrupole mass spectrometer. CID efficiency typically ranges from 40%-80%, although
it approaches 100% for some favorable cases (24). The amplitude of the rf signal that sets
the qg, value of the isolated ion during resonance excitation, termed the "tickle mass," must
be judiciously set as it will serve to eject all ions with m/z values below the tickle mass.
This limits the amount of low m/z fragmentation information obtained. A complete set of
complementary b- and y-type ions (25) is typically not obtained unless multiple stages of
mass spectrometry are performed. The g, value of the ion and the frequency and
amplitude of the tickle voltage must be carefully tuned to optimize fragmentation. The
auxiliary frequency generator outputs a single-frequency sinusoidal signal that is ‘not
sufficient to excite the envelope of ion signals resulting from isotopic abundances for ions
with large m/z values. Stored waveform inverse Fourier transform techniques (22) and the
application of random noise (26) have been successfully used to excite a broad range of ion
secular frequencies. In addition, shifting the g, value of the ion and increasing the
amplitude of the tickle pulse have substantially increased the amount of fragmentation
observed for large peptides (27).
53 —
2.2.6 High Resolution
The mass resolution of the ion trap mass spectrometer is a function of the number
of rf cycles that the ion spends interacting with the trapping field (28). Resolution is
increased by reducing the amplitude of the resonance ejection signal and reducing the
ejection scan speed, nominally 5555 u/sec for the Finnigan ITMS. The scan speed is
attenuated utilizing a network. of resistors placed in series with the digital-to-analog
converter (DAC) that controls the amplitude of the rf voltage applied to the ring electrode
(17). The fixed scanning rate of the DAC is applied to smaller "windows" of rf voltages
with a concomitant gain in the number of data points taken per unit mass. ‘A dc potential is
used as an offset to position the rf voltage, or mass window. This is schematically
illustrated in Figure 2.9. Figure 2.9 (a) shows the unattenuated mass window resulting
from scanning the amplitude of the rf voltage from 346 V to 7500 V while applying a
supplementary frequency at 120 kHz to extend the mass range by a factor of 3. This
increases the mass scan speed to 16665 u/s. Attenuation of the scan speed by a factor of
10 reduces the size of the mass window by the same factor, thus Figures 2.9 (b) - (d)
represent a 186 u mass window created by the attenuation. The different de offset voltages
serve to position the mass window in different regions within the mass window. In (b),
the mass window is positioned at 267 u, in (c) it is 800 u, and it is 1600 u in (d); therefore,
different regions of the mass window are accessed. Attenuation by a factor of 100 - 300 is
typically required to resolve the isotopes for singly to triply charged peptide ions to achieve
resolutions of 10,000 - 30,000 at m/z values ranging between 500 and 2000.
54
Figure 2.9: Extending the resolution on the quadrupole ion trap mass spectrometer. (a) A
normal resonance ejection scan from 90 u to 1950 u. (b) The scan speed is attenuated by a
factor of 10 resulting in a tenfold decrease in the width of the scanning mass window. The
dc offset is utilized to position the scanning window throughout the mass range. An offset
of 100 V positions the window at 267 u, (c) 300 V positions the window at 800 u, and (d)
600 V positions the window at 1600 u.
55 .
a) Scan Speed = 16,665 u/s
0 2000
b) Scan Speed = 1,666 u/s
DC offset = 100 V
—_——_
0. Scan Speed = 1,666 u/s 2000
Cc) DC offset = 300 V
a -
0 2000
d) Scan Speed = 1,666 u/s |
: DC offset = 600 V
0 2000
56
2.3 Comparison with Other Methods
As an ion storage device, an ion trap has the capability for high mass resolution,
mass range, sensitivity, and MS” that translates into versatile performance as a mass
spectrometer. In comparison to triple quadrupole and TOF mass spectrometers, the ion
| trap is unique in its ability to perform MS". All three techniques are about equal in terms
of mass accuracy and sensitivity. When utilizing electrospray ionization or MALDI, mid-
fmol to low pmol levels of sample are typically used to obtain both MS and MS/MS
spectra (29, 30), similar to results obtained by triple quadrupole and TOF mass
spectrometry. Lower levels are possible, but are not routine at the present. Sensitivity is
improved by varying the ion collection time and selectively injecting the ion of interest.
The ion trap, with MALDI, has shown equivalent performance to TOF mass
spectrometers at low mass range with the added advantage of exact precursor ion selection
and MS”. It is unlikely the ion trap will be as suitable for ultra-high mass analysis as TOF
mass spectrometers due to hardware limitations of the auxiliary frequency generator used
to extend the mass range. High molecular weight spectra obtained for singly-charged
proteins (30 - 50 kDa range) have shown results comparable to those obtained using a
linear time-of-flight (TOF) mass spectrometer without the implementation of delayed
extraction techniques.
There are several limitations of the performance of quadrupole ion trap mass
spectrometers. The alternate scan modes of triple quadrupole mass spectrometers such as
precursor ion and neutral loss scans are currently not possible. Furthermore, the number
57
of ions injected into the ion trap must be carefully controlled since space-charging can
degrade the performance of the instrument. This problem is solved through a rapid pre-
scan that assesses the ion current injected into the trap for ~50 ps, then sets the ionization
time to maximize the signal while minimizing space charge. Finally, when MS/MS is
performed, all ions with q, values below that of the resonance point will be ejected from
the ion trap; therefore, a complete sequence of complementary b- and y-type ions typically
cannot be obtained. Cotter et al. have recently shown that using low g, values in
conjunction with a heavier target gas affords full tandem mass spectra; consequently, the
ejection of low m/z fragment ions during CID is not a fundamental limitation of the ion
trap (31). Perhaps one major advantage of the ion trap not easily overlooked is the size of
the instrument. As lab space becomes tighter, the sizé of the ion trap and ease of
maintenance becomes a considerable advantage.
2.4 The New Generation of Ion Traps
In the past, the ITMS has not been an instrument well-suited for the robust and
routine analyses required by biochemists and biologists. High performance innovations to
the ITMS developed over the last several years have been used to build a new generation of
ion trap mass spectrometer, the Finnigan MAT LCQ. This instrument has been carefully
designed to interface with atmospheric pressure ionization techniques that are optimal for
the analysis of biomolecules. The operating characteristics of the instrument have been
changed by using a fundamental rf of 760 kHz instead of 1.1 MHz, an electrode spacing of
0.707 cm instead of 1.0 cm, and a q, value of 0.83 instead of 0.908 for resonance ejection
58
of ions (32). Ion injection into the ion trap has been optimized using a lensing system that
consists of two rf-only octopoles, resulting in a narrow spatial and energy distribution of
the injected ions (16, 33). Selective injection, trapping, and excitation of ions is performed
using tailored waveforms, analogous to the SWIFT technique (34). Unit mass resolution,
or the ability to separate an m/z value of 1500 from 1501, is maintained over the 2000
dalton mass range with a mass accuracy of 0.015% (35). These figures of merit are
comparable with the performance of current triple quadrupoles. It is expected that the mass
range of the LCQ will increase to 5000 daltons in the next year.
The most striking feature of the new ion trap is the software control of instrument
operation. Jon traps are operated through the use of a scan function that sets the ion
injection time, trapping voltages, cooling time, tickle voltages, and voltage ramping for
acquisition of the mass spectrum. Once a scan function is established for a ‘given
experiment, it can be used again but some parameters may need to be changed based on the
m/z value of the ion of interest. For example, MS, MS/MS, MS" and high resolution
experiments all require the construction of unique scan functions. Significant interaction
and expertise with the software was required with the older ion traps. The LCQ was
developed with Ion Trap instrument Control Language (ITCL), a computer language that
controls all of the elements of the scan function. For example, the hypothetical ITCL
command "hires 1200" would set up the scan function to isolate the ion at m/z 1200 then
slow the scan rate to achieve high mass resolution. All parameters required are
automatically set with the one ITCL command, compared with the necessity to manually
set a number of parameters using the ITMS software.
The ITCL language also enables the user to perform data-dependent experiments.
A mass scan can return to the computer program all the information it acquires during the
59
scan. For example, a command such as "hires mass( 1 )" would perform a high resolution
mass scan on the most intense ion returned from the previous mass scan. Very
complicated data-dependent routines such as “on-the-fly” tandem mass spectrometry can
be performed by stringing together commands in the form of a computer program. A
graphical user interface is employed to simplify the use of the ITCL language and to edit
the type of experiment desired during the course of an analysis. An m/z measurement,
followed by a high resolution scan to separate the isotopes of the desired ion for charge
state determination, followed by tandem mass spectrometry, is achieved by selecting the
experiment through the user interface. The software can automatically select precursor ions
based on some predefined criteria such as abundance, presence, or absence of an ion in a
predefined list. No user intervention in the process is required except for the initial setup of
the analysis. This level of control is unprecedented in mass spectrometry. In fact, the
reliance on embedded software control is so great that instrument upgrades will essentially
require downloading software from a CD-ROM to change operational parameters,
obviating the need for expensive additions of hardware, A number of different automated,
data-dependent experiments are possible including full range MS at unit resolution, MS”
with n=1 to 10, single ion monitoring (SIM) and single reaction monitoring (SRM), charge
state determination (utilizing the "ZoomScan") of up to +4 ions, and unit resolution
isolation up to m/z 1200.
2.5 Conclusion
The quadrupole ion trap is an extremely versatile instrument capable of performing
multiple stages of mass spectrometry with one mass analyzer. High resolution techniques
60
afford easy charge-state determination, facilitating the interpretation of data generated by
electrospray ionization. The sensitivity and performance characteristics of the instrument,
especially the automated experiments developed for the newly commercialized ion traps,
make quadrupole ion trap mass spectrometry an attractive technique to apply to the analysis
of biological and biochemical problems as is demonstrated in the following chapters.
61
2.6 References
1. McLuckey, S. A., Van Berkel, G. J., Goeringer, D. E., and Glish, G. L. (1994)
Anal. Chem. 66, 13, 689A-696A.
2. Dahl, D. A. (1995) , Lockheed Martin Idaho Technologies, Idaho Falls, ID.
3. March, R. E., and Hughes, R. J. (1989) Quadrupole Storage Mass Spectrometry,
(Winefordner, J.D. and Kolthoff, I.M., Eds.) in Chemical Analysis: A Series of
Monographs on Analytical Chemistry and its Applications, V102, Wiley & Sons, New
York.
4, | Paradisi, C., Todd, J. F. J., Traldi, P., and Vettori, U. (1992) Org. Mass Spectrom.
~27:251-254.
5. Paradisi, C., Todd, J. F. J., Traldi, P., and Vettori, U. (1992) Rapid Commun.
Mass Spectrom. 6 641-646. |
6. Williams, J. D., Reiser, H.-P., Kaiser, R. E., Jr., and Cooks, R. G. (1991) Int. J.
Mass Spectrom. Ion Proc. 108 199-219. |
7. Williams, J. D., Cox, K. A., Cooks, R. G., McLuckey, S. A., Hart, K. J., and
Goeringer, D. E. (1994) Anal. Chem. 66 725-729.
8. Guidugli, F., and Traldi, P. (1991) Rapid Commun. Mass Spectrom. 5 343-348.
9. Eades, D. M., Johnson, J. V., and Yost, R. A. (1993) J. Am. Soc. Mass Spectrom.
4917-929.
10. Cox, K. A., Williams, J. D., Cooks, R. G., and Kaiser, R. E., Jr. (1992) Biol.
Mass Spectrom. 21 226-241.
62
11. McLachlan, N. W. (1947) in Theory and Applications of Mathieu Functions,
Clarendon, Oxford.
12. Wuerker, R. F., Shelton, H., and Langmuir, R. V. (1959) J. Appl. Phys. 30 342-
349.
13. Soni, M. H., and Cooks, R. G. (1994) Anal. Chem. 66 2488-2496.
14. McLuckey, S. A., Goeringer, D. E., and Glish, G. L. (1991) J. Am. Soc. Mass
Spectrom. 2 11-21.
15. Doroshenko, V. M., and Cotter, R. J. (1993) Rapid. Commun. Mass Spectrom. 7
822-827.
16. Jonscher, K. R., and Yates, J. R., IIT (1996) Anal. Chem. 68 659-667.
17. Kaiser, R. E., Jr., Cooks, R. G., Stafford, G. C., Jr., Syka, J. E. P., and
Hemberger, P. H. (1991) Int. J. Mass Spectrom. Ion Proc. 106 79-115.
18. Dawson, P. H., Hedman, J., and Whetten, N. R. (1969) Rev. Sci. Instrum. 40
1444-1450.
19. Mather, R. E., Waldren, R. M., and Todd, J. F. J. (1978) Dyn. Mass Spectrom. 5
71-85.
20. Louris, J. N., Cooks, R. G., Syka, J. E. P., Kelley, P. E., Stafford, G. C., Jr., and
Todd, J. F. J. (1987) Anal. Chem. 59 1677-1685.
21. Kaiser, R. E., Jr., Louris, J. N., Amy, J. W., and Cooks, R. G. (1989) Rapid
. Commun. Mass Spectrom. 3 225-229.
22. Julian, R. K. (1993) Anal. Chem. 65 1827-1833.
23. Cooks, R. G., McLuckey, S. A., and Kaiser, R. E., Jr. (1991) Chem. Eng. News
69 26-41.
63
24. Cooks, R. G., and Kaiser, R. E., Jr. (1990) Acc. Chem. Res. 23 213-219.
25. Roepstorff, P., and Fohiman, J. (1984) Biomed. Mass Spectrom. 11 601.
26. McLuckey, S. A., Goehringer, D. E., and Glish, G. L. (1992) Anal. Chem. 64
1455-1460.
27. Qin, J., and Chait, B. T. (1995) Proc. of the 43rd ASMS Conf. on Mass Spectrom.
and Allied Topics May 21-26, Atlanta, GA, American Society for Mass Spectrometry, p.
1100.
28. Fischer, E. (1959) Z. Phys. 156 1-5.
29. Yates, N. A., Shabanowitz, J., and Hunt, D. F. (1994) Abs. Paps. Am. Chem. Soc.
208 132-ANYL.
30. Qin, J., and Chait, B. T. (1995) Proc. of the 43rd ASMS Conf. on Mass Spectrom.
and Allied Topics, May 21-26, Atlanta, GA, American Society for Mass Spectrometry, p.
989.
31. Cotter, R. (1996) Photonics West Conference, Jan. 27-Feb. 3.
32. Schwartz, J. C., Bier, M. E., Taylor, D. M., Zhou, J., Syka, J. E. P., James, M. S.,
and Stafford, G. C. (1995) Proc. of the 43rd ASMS Conf. on Mass Spectrom. and Allied.
Topics, May 21-26, Atlanta, GA, American Society for Mass Spectrometry, p. 1114.
33. Bier, M. E., Schwartz, J. C., Zhou, J., Taylor, D., Syka, J., James, M., Fies, B.,
and Stafford, G. (1995) Proc. of the 43rd ASMS Conf. on Mass Spectrom. and Allied
Topics, May 21-26, Atlanta, GA, American Society for Mass Spectrometry, p. 1117.
34. Taylor, D., Schwartz, J., Zhou, J., James, M., Bier, M., Korsak, A., and Stafford,
G. (1995) Proc. of the 43rd ASMS Conf. on Mass Spectrom. and Allied Topics, May 21-
26, Atlanta, GA, American Society for Mass Spectrometry, p. 1103.
64
35. Land, A. P., Wheeler, K., Mylchreest, I. C., Sanders, M., and Jardine, I. (1995)
Proc. of the 43rd ASMS Conf. on Mass Spectrom. and Allied Topics, May 21-26, Atlanta,
GA, American Society for Mass Spectrometry, p. 653.
65
Chapter 3
Matrix-Assisted Laser Desorption of Peptides and Proteins on
a Quadrupole Ion Trap Mass Spectrometer
3.1 Overview
The use of ultraviolet matrix-assisted laser desorption ionization (MALDID) to ionize
peptides and proteins for analysis in a quadrupole ion trap is described. An ion source was
modified to accommodate a fiber optic to transmit laser radiation from a nitrogen laser
(337 nm) to the tip of the sample probe containing peptide or protein samples in a matrix
of 2, 5-dihydroxy benzoic acid (DHB) or sinapinic acid. Detection limits are demonstrated
with 10 fmol of sperm-whale myoglobin. The dimer of sperm-whale myoglobin was also
observed at m/z 34,430. A comparison is made between the tandem mass spectrum
(MS/MS) of human angiotensin I desorbed by MALDI and the mass spectrum for the
peptide desorbed by liquid secondary ion mass spectrometry. Both spectra were found to
_ contain abundant structural information.
In a practical application of the technique, the dominant phosphorylation site of the
P protein from Sendai virus is localized by MALDI/quadrupole ion trap mass
spectrometry. The P protein from Sendai virus, a murine paramyxovirus, is reported in the
literature to be a highly phosphorylated protein. In vitro studies have detected
phosphorylation in different regions of the protein while a single phosphopeptide was
66
observed using in vivo techniques. Analysis by mass spectrometry revealed two
phosphopeptides proximal in the P protein sequence.
3.2 Instrument Development
3.2.1 Introduction
Ultraviolet matrix-assisted laser desorption ionization (MALDI) has been an
effective means of creating ions for the molecular weight analysis of large biomolecules (1-
4) and complex mixtures of peptides and proteins (5). The pulsed nature of the ionization
event and the high velocities of the resulting ions has limited its use mainly to time-of-
flight mass spectrometers, although this technique has been applied more recently to
magnetic sector instruments with integrating array detectors (6, 7) and ion trap mass
spectrometers (8-13). Instruments such as the Fourier-transform mass spectrometer
(FTMS, FT-ICR) and the quadrupole ion trap mass spectrometer (ITMS) function as ion
storage devices and hence are well-suited for use with pulsed ionization techniques. .
Additionally, these instruments are capable of analyzing small quantities of sample and
performing multi-stage tandem mass spectrometry (MS") experiments (14-16). These
features, in conjunction with the utility of MALDI for the ionization of peptides and
proteins present in complex mixtures, create a powerful approach for structural
characterization of peptides and proteins.
Interfacing MALDI to ion trap mass spectrometers presents a unique challenge.
Ions are created with high velocities and wide angular distributions which may be difficult
to trap in the mass analyzer (17). Two approaches have been used in FTMS instruments to
67
trap these ions. The first approach takes advantage of collisional damping to trap the
desorbed ions. Buchanan and co-workers pulsed argon into the ion cyclotron resonance |
(ICR) cell concurrent with the laser pulse to damp the velocity of the ions to a level where
they could be trapped in the magnetic field (8, 9). Mass analysis was delayed until the
argon was pumped from the ICR cell to improve mass resolution. Russell and co-workers
employed a laser desorption ion source recessed slightly from the entrance of the ICR cell
which was designed to allow a small volume of helium to be pulsed into the source
concurrent with the laser radiation (12). The velocity of the desorbed ions was damped as
they collided with helium in the source and then drifted from the source into the ICR cell.
A second approach utilized by Wilkins and co-workers created a potential well between the
trapping plates to constrain the desorbed ions once they entered the ICR cell (10). Proteins
as large as 34,000 Da have been ionized, trapped, and analyzed by this method.
The trapping of ions in quadrupole ion traps is less problematic since the ion trap is
operated with ~1 mtorr of helium in the trap volume. Ions injected into or created in the
trap lose kinetic energy as they undergo collisions with the helium (18). Two approaches
' utilizing laser desorption have been used to introduce ions into the ion trap. In the first,
employed by Louris and co-workers, ions were generated near the endcap aperture then
| extracted and focussed axially into the ion trap (19). Rather than transmit the laser radiation
through the cavity of the trap, the sample probe was recessed from the entrance aperture
and the probe tip irradiated by a fiber optic at an angle of ~90° relative to the ion-optical
path. Ions were extracted and focused into the entrance endcap aperture using an einzel
lens. A second approach involved radial desorption of ions into the cavity of the ion trap
through a hole in the ring electrode (20-22). In this case two holes were drilled on opposite
sides of the electrode, one for insertion of the sample probe into the wall of the ring
68
electrode and the other for transmitting the laser radiation to the sample probe. Ions were
then desorbed directly into the cavity of the ion trap. The above two methods were
pioneered with non-matrix assisted laser desorption techniques.
Cox and co-workers demonstrated MALDI using an arrangement similar to that of
Louris and co-workers and successfully observed bovine insulin B-chain (11). Chambers
and co-workers obtained a mass spectrum of horse cytochrome c using a MALDI
arrangement as described in Ref. 21 (13). Both of these examples illustrate that MALDI
experiments can be conducted on ITMS instruments.
The objectives of this study were to extend the earlier work of Cox and co-workers,
to determine if an external ionization source on a quadrupole ion trap can be used to focus
protein ions created by MALDI into the ion trap, and to obtain an estimate of the detection
limits of the technique. In addition, we examined the feasibility of performing MS/MS
experiments on peptide ions generated from MALDI to determine the potential for
extending protein sequence analysis to MALDI-ITMS. A preliminary account of this
work has been presented (23). At the same conference, Bier and co-workers also
presented a preliminary account of peptide and protein analysis using MALDI-ITMS with
mass range extension by reducing the fundamental rf frequency to extend the nominal
mass range of the ion trap (24).
3.2.2 Experimental
3.2.2.1 Ionization Source
The external MALDI source was constructed of modified components from a
Finnigan MAT continuous-flow fast atom bombardment ion source (Bioprobe source
69
block, Finnigan MAT, San Jose, CA, USA) to improve positioning of the fiber optic. The
sample probe was electrically isolated from the ion source block with a Vespel sleeve to
allow the application of an independent potential to the probe. In general the probe was
kept near ground potential. Lenses 1 and 2 were modified by drilling the aperture to a
diameter of 0.150" to afford a wider angle of ion acceptance into the optical path. The
diameter of lens 3 was 0.125". The sample probe was approximately 0.100" from the first
lens. Typical voltages for the lenses were as follow: lens 1 -2 V, lens 2 -196 V, lens 3 -24
V, and the ion trap was floated at -9 V. An ion gating lens was positioned prior to the
entrance aperture of the endcap electrode and its potential was varied between +36 V (gate
closed) and -186 V (gate open). The helium pressure in the mass analyzer region was
nominally 1 mTorr.
The 200 tm fiber optic (Radiant Communications Corporation, South Plainfield,
NJ, USA) was passed into the vaccum manifold through a 1/8" diameter inlet on a custom
vaccum flange (MDC, Hayward, CA, USA). A hole was drilled through lens 1 to position
the optical fiber so that the transmitted radiation impinged upon the probe tip at an angle of
approximately 45°. The fiber optic was coupled to a VSL-337 nitrogen laser (Laser
Science, Inc., Newton, MA, USA) using a single-mode adapter. The laser was rated at 3
mW maximum average power and 175 uJ/pulse maximum energy (at 10 Hz). A 200 pm
or 400 [1m optical fiber was employed to transmit the beam. The laser power exiting the
fiber optic was estimated to be 3.7 x 10’ W/cm’ for the 200 um fiber and 1.45 x 10’
W/cm’ for the 400 tm fiber. These estimates are based on coupling efficiencies of 20%
(200 um) and 40% (400 pm fiber). Laser repetition rates were typically ~4.5 Hz (220
ms/cycle) for single-stage mass spectrum scans and ~3.7 Hz (270 ms/cycle) for MS/MS
scans and were adjusted by entering a delay in the ITMS scan function. A laser pulse was
70
initiated from the TTL signal used to trigger the ion gate on the ITMS electronics. A diode
and emitter-follower were used to protect the gate signal, while an RC delay was placed
between two non-inverting CMOS hex buffers to obtain a TTL signal to trigger the laser
~500 us after the rise of the gate.
3.2.2.2 Mass Spectrometry
A quadrupole ion trap using components of an ITMS (Finnigan MAT) and a triple
quadrupole mass spectrometer, TSQ 70 (Finnigan MAT), was constructed at Finnigan
MAT. The ITMS was housed in the differentially pumped region of the TSQ 70 vacuum
manifold facilitating the coupling of commercially available TSQ 70 ion sources to the ion
trap. A 3 kV lens was placed near the exit endcap electrode to focus ions to the 20 kV
conversion dynode/electron multiplier assembly. The rf signal from the ITMS electronics
was directed onto the ring electrode via a ceramic feedthrough in a central flange in the
vacuum manifold. The signal from the electron multiplier was transferred to the ITMS
electronics and the resulting mass spectrum displayed on a Compaq 386 computer. The
auxiliary frequency generator in the ITMS electronics was used to place a supplementary rf
signal on the endcap electrodes. Typically, 6 microscans were summed in the memory of
the Compaq 386 computer and transferred to a DECStation 2100 workstation where each
set of scans were further averaged using modified ICIS software. A 2-6 point mass
calibration curve was generated with the software (created by Joe Zhou at Finnigan MAT)
to display calibrated spectra. Calibration curves were stable for several days if conditions
were not altered (e.g., mass range and helium pressure).
71
An example of a single-stage mass spectrum ion trap scan function is shown in
Figure 3.1(a) and typically included a 100 ms delay to adjust the laser cycle, followed by a
5 ms ionization period during which ions were injected into the ion trap. During ion
injection the amplitude (700-3500 Vow) of the fundamental rf was chosen to optimize
signal intensity for the ion of interest. Typically, the rf amplitude was increased: to a
constant value for 6 ms to eject low mass matrix ions and to reduce space charge effects.
The conversion dynode and electron multiplier were set at -15 kV and 1.7 kV, respectively
for data acquistion.
The ITMS scan function used to record an MS/MS spectrum is shown in Figure
3.1(b) and typically included a 50 ms cycle time delay, 5 ms ionization period, and 6 ms
ejection pulse. A precursor ion was isolated with a 30 ms reverse scan with resonance —
ejection applied to remove ions with mass-to-charge ratios higher than that of the ion of
interest and a 30 ms forward scan to eject ions of mass-to-charge ratios lower than that of
the ion of interest (25, 26). The ion was then brought into resonance at a q, value of 0.2-
0.3 with a small supplementary rf voltage (0.5-2.5 V) which was applied for 30 ms to
promote fragmentation via collisions with the helium bath gas. The mass spectrum
resulting from ‘the fragmentation products was then acquired using the appropriate mass
range.
. 3.2.2.3 Sample Preparation
Porcine renin substrate tetradecapeptide (1759 Da), human angiotensin I (1296 Da),
bovine insulin (5734 Da), sperm-whale myoglobin (17,199 Da), and porcine elastase
(25,898 Da) were purchased from Sigma Chemical Company (Saint Louis, MO, USA)
72
Figure 3.1: (a) ITMS scan function for a molecular weight scan. (b) ITMS scan function
for an MS/MS scan. MS/MS is accomplished by isolating the ion of interest with a
combination of forward and reverse scans, then inducing collision-induced dissociation via
resonance excitation.
73
a) MS Scan Function’ c
_ d e
rf amplitude |
a . |
500 Lsec— > <7 —
| ag ! L200 msec +
laser trigger | [ |
a- delay to adjust laser cycle
b- ionization period
c- pulse to eject low mass ions ©
d- turn on multiplier
e- acquisition
b) MS/MS Scan Function
rf amplitude
— 500 sec > = .
~— ! ‘ ~250 msec |
laser trigger ! | | |
a - delay to adjust laser cycles e- forward scan to eject ions
b - ionization period , below m/z of interest
c - pulse to eject low mass matrix ions f - "tickle"
d- reverse scan to eject ions above m/z of interest’ = g- turn on multiplier
h- acquisition
74
and used without further purification. 2,5-Dihydroxybenzoic acid (DHB) and sinapinic
acid were purchased from Aldrich Chemical Company (Milwaukee, WI, USA). Sample
and matrix were dissolved in a 1:1 mixture of ultrapure water and high-performance liquid
chromatography (HPLC) grade acetonitrile (EM Science, Gibbstown, NJ, USA). Sample
stock solutions were at a concentration of ~1 nmol/uL with matrix solutions at ~1000-
2000 nmol/L. Spectra were acquired using equivolume mixtures of sample and matrix
giving a 1:1,000 sample-to-matrix ratio deposited onto a 0.050" stainless steel probe tip.
Sample dilutuions for detection limit experiments were carried out on a polished Teflon
plate and aliquots of sample and matrix solutions were mixed to obtain the desired
concentration.
3.2.3 Results and Discussion
MALDI is a versatile technique for the ionization of peptides and proteins. In light
of the utility of this ionization method, it was of interest to couple a MALDI source to a
quadrupole ion trap mass spectrometer for the analyses of peptides and proteins. Previous
research on a MALDI-ITMS using a fiber optic to transmit laser radiation has
demonstrated the ability to analyze molecules up to 3500 Da in molecular weight (27). The
analysis of larger peptides or proteins by this approach has recently been reported by
Chambers and co-workers; however, they were unable to observe ions with values of m/z
greater than 13,000 Da and realized a significant drop in sensitivity above m/z 3000 (13).
Here we report the mass analysis of proteins of molecular weight up to 26 kDa by
75
MALDI-ITMS using an external ion source and fiber optic transmission of radiation from
a nitrogen laser.
Initial MALDI experiments utilized an ion source configuration similar to that of
Wright and co-workers (28). In this experiment the laser beam was directed orthogonal to
the sample probe and intercepted the probe surface at an angle of 45°. The intensity of the
ion signals observed with this method were weak (signal-to-noise ratio 3:1) and sporadic.
In laser desorption experiments using a UV absorbing matrix, Beavis and Chait noted that
the ion plume is directed normal to the probe surface and that the ion velocity is 750 m/sec
(17). This would account for the relatively weak ion signals since it is unlikely that the
lensing system would be able to redirect the initial 45° trajectories of the fast moving ions
into the ion optical path. The ion source was then modified to place the probe surface
perpendicular to the ion optical path and to position the fiber optic at an angle of ~45°
relative to the probe surface normal. This configuration is depicted in Figure 3.2.
Shown in Figure 3.3 is the mass spectrum of the tetradecapeptide, renin substrate,
obtained by desorbing a 1:2000 mixture of 250 fmol of peptide and DHB applied to the
sample probe. All ions below 95 u had unstable trajectories during injection and were
ejected from the trap. In this manner the effects of space charge were reduced by limiting
the number of low-mass matrix ions in the trap. Peptide ions were resonantly ejected with
an auxiliary field frequency of 89,202 Hz having 8.8 V,, amplitude. To determine the
reproducibility of mass measurements and resolution, a total of 10 mass spectra were
acquired from the 250 fmol sample. Mass resolution and mass measurement accuracy
averaged 300 (measured as full width at half height (FWHH)) and 0.082%, respectively.
For the analysis of proteins, the fiber optic was switched to a 400 um diameter
fiber to increase the area of irradiation. The laser power density based on estimated
76
Figure 3.2: Schematic diagram of the MALDI-QITMS system. The ion trap electrodes
are located in the analyzer region of the differentially pumped vacuum manifold of a TSQ
700 triple quadrupole mass spectrometer.
77
roy dnnyy uonse[q J
. F-
sux]
3pouAC] UOTSIOAUOZ) |
dei, uoy
sondg uoy
eqolg ajdures
Jase’] UasOIIN wu /¢¢ OF,
petdnoD seqny Teondo
78
Figure 3.3: Mass spectrum of 250 fmol of tetradecapeptide renin substrate in a 1:2000
ratio with 2,5-dihydroxybenzoic acid. [M+H]t = 1758.9 (average). The ‘exclusion limit
was 95 u and resonance ejection occurred at a frequency of 89,202 Hz, 8.8 V,-p amplitude.
80
Relative Abundance
100
60
40
20
1500
1600
79
(M+H)*
1759.0
i | AT
1700. | 1800
m/z
1900
2000
80
coupling efficiencies provided by the manufacturer show the laser power of the 400 um
fiber optic to be less than that of the 200 pm fiber optic (by roughly 3-fold) but still within
the range necessary for matrix-assisted laser desorption (10’ W/cm’). Shown in Figures
3.4(a) and 3.4(b) are the mass spectra of bovine insulin and cytochrome c desorbed using a
400 jm fiber optic. The mass spectra result from 60 and 114 laser shots, respectively,
with each spectrum exhibiting mass resolution of approximately 100. In both examples
ions below m/z 300 were excluded from the trap. Some fragmentation may have occurred,
indicated by the appearance of ions characteristic of insulin B chain. The mass resolutions
calculated were comparable to those observed on time-of-flight instruments due, in part, to
the increased scan rate associated with mass range extension using resonant ejection.
Schwartz and co-workers have observed that mass resolution can be increased dramatically
with a slowing of the scan speed by keeping ions near resonance for a longer period of
time prior to ejection from the ion trap (29). In these experiments no attempts were made
to increase the mass resolution by decreasing the scan speed.
3.2.3.1 Tandem Mass Spectrometry
Multiple-stage mass spectrometry performed on the ion trap has been demonstrated
by a number of groups (14-16, 20). Ions in the trap may be resonantly excited by the
application of a supplementary rf voltage on the endcap electrodes corresponding to the
secular frequency of the precursor ion. The amplitude of the supplementary rf must be
chosen to optimize fragmentations due to collisions with the helium bath gas without
ejecting the ions corresponding to the protonated molecule. The amplitude of the rf voltage
81
Figure 3.4: (a) Bovine insulin in 2,5-dihydroxybenzoic acid at a ratio of 1:1000. Ions
below 300 u were not trapped and ions were ejected at 23,351 Hz, 5.2 V.... [M+H]* =
5707 (average). (b) Bovine cytochrome c in a 1:1000 mixture with sinapinic acid.
[M+H]t = 12223 (average). The exclusion limit was 330 u and ions were resonantly
ejected at 7,848 Hz, 9 Vp"
Relative Abundance
82
M+H)*
100; 3) (M+H)
804
60:
40: .
b-chain
201
3000 4000. 5000 6000 7000 8000
(M+H)*
100) b)
- 80:
60:
40:
/ call Ntdyunlh
6000 8000 10000 12000 14000
m/z
83
is experimentally determined. Kaiser and co-workers demonstrated MS/MS of peptide
ions produced from liquid secondary ion mass spectrometry (LSIMS) (14). The mass
spectrum was rich in structural information showing abundant b- and y-type ions. Shown
in Figure 3.5 are the MS/MS spectra of angiotensin I produced by peptides
desorbed/ionized by (a) MALDI (in a 1:1000 ratio with DHB) and (b) LSIMS (in a
thioglycerol matrix) using resonance excitation at a g, of 0.3. All ions with m/z values
below 430 u were ejected during excitation. The excitation frequency for the MALDI
spectrum was 120,632 Hz at an amplitude of 3.18 Ve while that for the LSIMS spectrum
was 121,552 Hz at an amplitude of 3.10 Ve In both examples 100 scans were averaged
representing 600 laser or Cst ion pulses. The mass spectra are nearly identical. The
predominant fragment ions observed in the spectra are b- and y-type ions. Signals resulting
from neutral losses of water, ammonia, and carbon monoxide (a-type ions) are also
present. The peak marked with an asterisk is thought to result from surface interactions.
The extent of fragmentation observed in the MS/MS spectrum is encouraging since
fragment ions are observed for all amide bonds in the peptide except one.
3.2.3.2 Trapping large protein ions: effect of ionization exclusion limit
At a given ion kinetic energy, the exclusion limit specifies the ion of lowest m/z that
can maintain a stable trajectory within the ion trap. Theoretically, at a given kinetic energy,
the optimal rf voltage is proportional to the square root of the mass of the injected ion (27)
Zz,
JKE-m (Eq. 3.1)
Vv, =
84
Figure 3.5: (a)) MALDI-MS/MS spectrum of angiotensin I in 2,5-dihydroxybenzoic acid
at a 1:1000 ratio. The precursor ion was resonantly excited at 120,632 Hz, 3.18 V,.. (b)
LSIMS/MS spectrum of angiotensin I in a thioglycerol matrix. The precursor ion was
excited at 121,552 Hz, 3.10 V,... Both spectra result from 600 laser/Cs* shots. In both
cases, fragment ions are observed for all but one amide linkage.
Relative Abundance
85
DRVYIHPFAL
100) a) x8| % x1|
10
807.
607
401.
| Y, 0 *
. La
400 600 800 1000 1200 1400
m/z
DRVYIHPFHL
00,;...~~C~C
10
807
Salk
400 600 800
1000 1200 1400
m/z
86
where V,, = optimal rf amplitude, z,= distance from the center of the trap to the endcap
vertex, and @ = fundamental frequency (1.1 MHz). Kaiser, using CsI cluster ions
generated by LSIMS, showed that the exclusion limit is linear with respect to the square
root of the mass of the injected ions over a large mass range (<6000 Da) (27). The range
of optimal rf voltages used to trap an ion of specific mass and energy is generally narrow
and may cause significant mass discrimination. To estimate the conditions for trapping
large ions generated by MALDI, optimal rf voltages were determined for a variety of
peptide ions generated by LSIMS, ranging in m/z from 1295 (angiotensin I) to 3500
(bovine insulin B chain). The experimental optimal rf voltage expressed as an exclusion
limit was found to be proportional to the square root of the mass of the injected ion as.
predicted by theory. In addition, these conditions were found to be suitable for trapping
ions generated by MALDI. Figure 3.6 is a graph of the exclusion limits used to trap
MALDI ions vs. the square root of the mass of the injected ion. A linear relationship is
found over a large mass range and therefore this relationship can be used to predict the
exclusion limit necessary to trap ions of a desired m/z.
Shown in Figure 3.7 is the mass spectrum of a mixture of whale myoglobin
(17,199 Da) and porcine elastase (25,898 Da). The dimer of myoglobin (34,399 Da) also
appears. This experiment illustrates that proteins with a molecular weight difference of at
least 17 kDa may be trapped and mass analyzed under the same set of conditions.
However, this does not imply that the conditions were optimal for all the ions and some
mass discrimination may have occurred. The exclusion limit of 430 u was chosen to
optimize trapping efficiency for the dimer of myoglobin. The mass range was extended to
40,000 u using a resonance signal at 5883 Hz, with 5 Vp amplitude. These conditions
87
Figure 3.6: Optimal rf voltage expressed as an exclusion limit vs. square root of mass.
The relationship appears to be linear throughout the mass range.
exclusion limit
500
400
300
200
100
88
9 myoglobin dimer —
& elastase
© trypsin
myoglobin
insulin
angiotensin
I —_ a |
100 200
Square root of mass
89
Figure 3.7: Mass spectrum of 50 pmol porcine elastase, [M+H]* = 25,871 (average), and
100 pmol of myoglobin, [M+H]* = 17,143 (average). The dimer of myoglobin appears
at [M+H]*+ = 34,430 (average). The exclusion limit was 430 amu and ions were ejected
using a signal at 5883 Hz, 5 V,,..
Relative Abundance
100}
80)
60 |
AQ"
20 |
(M+H)t
myoglobin
90
(M+H)*t
elastase
(2M+H)*
10000. 15000. 20000 25000 30000 35000 40000
m/z
91
resulted in a mass resolution of 31 and 85 for elastase and the dimer, respectively. Proteins
of higher molecular weight should be observable by reducing the fundamental rf frequency
to extend the nominal mass range of the ion trap (24).
3.2.3.3 Detection Limits
The detection limit of a mass spectrometer is a good indicator of the utility of the
system for a given application. Protein ions generated by MALDI from 1 fmol of
cytochrome c and 700 fmol of a B-galactosidase subunit (MW 116 kDa) have been
observed on a time-of-flight instrument (20, 30). Additionally, 2.1 amol of a peptide,
gramicidin S, has been observed on an ITMS interfaced with LSIMS (14). Thus, the
potential for a high sensitivity technique based on a MALDI and ITMS combination for the
analysis of peptides and proteins is clearly evident. To test the system’s ability to detect
low quantities of sample, successively smaller amounts of sample and matrix were applied
to the sample probe and analyzed. To insure that no sample was carried over between each
analysis, the probe tip was filed down, soaked in 5% acetic acid, and sonicated in methanol
for 10 min. A matrix blank was used to verify that no sample remained on the probe tip.
Analyses of the protein myoglobin using 500 to 0.1 fmol of the sample applied to the
probe was attempted. Below 10 femtomoles ions were observed, but the signal was
sporadic. Stable signal was observed at the 10 fmol level.
Shown in Figure 3.8 is the mass spectrum for 10 fmol of myoglobin. A total of 10
scans were averaged from 60 laser shots and a mass resolution of 50 with a signal-to-noise
ratio of 5:1 was observed. As has been noted in the literature, the local homogeneity of the
92
Figure 3.8: 10 fmol of whale myoglobin in sinapinic acid at a 1:11 x 10° ratio. [M+H]*
= 17,294 (average). The exclusion limit was 330 u and resonance ejection occurred at
7848 Hz, 9 V,_. The mass spectrum results from averaging 10 scans.
Relative Abundance
100 }
80 7
60 7
2018
93
(M+Hy
(M+2H3
so00 10000 12000 14000 16000 18000 20000
m/z
94 -
matrix/analyte solution is an important parameter for successful MALDI-MS, as is the
analyte:matrix ratio (31, 32). Optimization of the sample preparation and deposition may
produce more consistent signal at lower sample levels.
3.3 Application of MALDI/ITMS for Analysis of Phosphopeptides
3.3.1 Overview
The P protein from Sendai virus, a murine paramyxovirus, is reported in the
literature to be a highly phosphorylated protein. In vitro studies have detected
phosphorylation in different regions of the protein while a single phosphopeptide, identified
as the sole phosphorylation site, was observed using in vivo techniques. In this work,
using a direct approach, two phosphorylation sites of the P protein from Sendai virus are
localized by matrix-assisted laser desorption ionization (MALDI)/quadrupole ion trap mass
spectrometry. A computer-aided approach is used to confirm peptide identification.
3.3.2 Introduction
Sendai virus is the murine prototype of the paramyxovirus family, belonging to the
order of Mononegalvirales. Related human pathogens include a number of types of
parainfluenza virus, mumps, measles, and respiratory syncytial virus; as well as the more
distantly related filoviruses, Marburg and Ebola. Animal pathogens comprise Newcastle
disease, cattle and bird parainfluenza viruses, canine distemper, and murine pneumonia.
95
An understanding of the functioning of the Sendai virus prototype would provide insights
into the function and replication of these ubiquitous human and animal viruses.
The genome of the Sendai virus consists of a single-strand of RNA with negative
polarity that codes for at least six structural and five non-structural proteins (33-35). The
RNA core is encapsidated by a helical nucleocapsid protein (NP). Virions enter cells
directly through surface membranes, and viral replication and transcription, mediated by
viral polymerase, begins immediately in the cytoplasm. Polymerase activity is carried out
by the polymerase-associated phosphoprotein, P protein, of MW 65,000 u and the large
protein, L protein, of MW 200,000 u (36, 37). Almost all of the viral proteins are
phosphorylated; however, the P protein appears to be more heavily phosphorylated on a
mole-per-mole basis (38, 39). The P protein also seems to be modular in nature (40). _N-
terminal and C-terminal domains are conserved among paramyxoviruses (52% and 69%
homology, respectively) while a 100 residue region in the middle is variable with ~11%
homology (41, 42). The C-terminal domain has been shown to stabilize the L protein (43)
and the N-terminal region interacts with the NP protein and has been shown to be essential
for RNA encapsidation as well as RNA synthesis (44).
A number of studies were undertaken to locate sites of post-translational
modification to understand the role phosphorylation plays in the function of the P protein.
In vitro experiments reported conflicting results where phosphorylation sites were detected
in the first N-terminal quarter of the protein (39) or in the second N-terminal quarter of the
protein (45). More recent work (46) showed that cell-free phosphorylation using virion-
associated protein kinase (VAPK) as a phosphorylating agent caused phosphorylation of
both serine and threonine. In contrast, intracellular experiments in which the
phosphorylation state of the P protein was analyzed during virus replication indicated that
96
phosphorylation occurred only on serine residues. The number of detected
phosphorylation sites also differed ‘between the in vitro and the in vivo techniques.
Enzymatic digestion of the VAPK-phosphorylated P protein using trypsin, followed by
two-dimensional thin layer electrophoresis (2D TLE), produced four major spots and nine
minor spots (46). Similar experiments utilizing intracellular analysis produced one major
spot with several minot spots and it was concluded that the P protein in infected cells was
primarily phosphorylated at one or a set of adjacent sites (46). Site-directed mutagenesis
was subsequently used to identify the primary location of phosphorylation on the P protein
(47). In a parallel effort described here, direct analysis using quadrupole ion trap mass
spectrometry was employed to identify P protein phosphorylation sites.
3.3.3 Experimental
3.3.3.1 Mass Spectrometry
A quadrupole ion trap mass spectrometer (ITMS) (Finnigan MAT, San Jose, CA,
USA) placed in the vacuum manifold of a TSQ 70 triple quadrupole mass spectrometer
(Finnigan MAT) was interfaced to an external matrix-assisted laser desorption ionization
(MALDI) source as described previously (48). A 200 um core fused silica optical fiber
was used to ionize the sample by irradiation with a 337 nm beam from a nitrogen laser
(Laser Science, Inc., Newton, MA, USA). The ion trap volume was filled with helium to
an uncorrected pressure of 5 x 10% Torr, then argon was added using a separate needle
valve to bring the final uncorrected pressure to 5.5 x 10* Torr. The addition of ~10%
(pressure/pressure) argon to the trapping volume serves to improve the trapping efficiency
(49), the resolution of the mass spectrum, and slightly increases the amount of
97
fragmentation achieved upon injection into an ion trap. The ion beam was focused into the
ion trap using a three-element einzel lens. Typical lens voltages were as follows: Lens 1
-5 V, Lens 2 -190 V, Lens 3 -20 V, Ion gate -200 V (open)/+ 200 V (closed), trap float
-11 V, and the probe was held at 5 V.
The ion trap scan employed to obtain a single-stage mass spectrum consisted of
the following steps (48): A 100 ms delay was used to adjust the laser cycle to ~4.5 Hz.
Ions were allowed into the ion trap during a 5 ms ionization period when the gating tube
lens was set to “open.” The TTL signal used to trigger the ion gate operation was also
employed to trigger the laser to fire ~500 ls after the ion gate opened. The rf level during
the ionization period was typically set to 1154 V,,. This enabled ions with m/z values
above 100 u to be stably trapped. The amplitude of the rf voltage was ramped to 7500 Vj,
in order to eject matrix ions with m/z values less than 650 u. The electron multiplier was
then turned on. Data acquisition was accomplished by ramping the amplitude of the rf
signal while applying a supplementary signal to the endcap electrodes to resonantly eject
ions through the endcap electrode and into the conversion dynode/electron multiplier
detection system (25, 50). Details appear in the relevant figure captions. The conversion
dynode and electron multiplier were set at -15 kV and 1.2 kV, respectively, for data
acquisition. Supplemental signals were also applied to the endcap electrodes to cause
amplification of ion trajectories by resonance excitation (14). Subsequent ion
fragmentation was induced by collisions with the helium damping gas and afforded amino
acid sequence information. Mass spectra were displayed on a Compaq 386 PC then ported
to a DECStation 2100 for data acquisition and analysis. Baseline subtraction at a given
threshhold was performed to remove artifacts due to software normalization.
98
A LaserMAT (Finnigan MAT) linear time-of-flight (TOR) instrument was also
‘used to screen fractions for the presence of phosphopeptides and to determine the number
of peptides in each sample. Ions generated by the MALDI process were extracted from the
source and accelerated into a field-free flight tube employing a 20 kV potential. Detection
was accomplished using a 15 kV conversion dynode coupled to an electron multiplier.
3.3.3.2 Sample Preparation
Recombinant ”P labeled P protein from Sendai virus was infected into CV1 cells
and purified by immunoprecipitation (47) then enzymatically digested with trypsin or
chymotrypsin. A portion of the protein digests were separated by 2D TLE. Spots on the
gel were excised and peptides extracted (46). Aliquots of both the protein digests and the
extracted peptides, estimated to contain 1-3 nmol of material, were lyophilized and
reconstituted in 0.1% trifluoroacetic acid (TFA) (Aldrich Chemical Co., Milwaukee, WI,
USA) to aconcentration of 0.1 - 1 mM. This material served as a stock solution and was
stored at -4° C.
3.3.3.2.1 Chromatography
Aliquots of the stock solutions representing an. estimated 250 pmol of material
were loaded onto a 20 WL Peek injection loop and concentrated by washing with 100%
Solvent A (0.1% TFA) on an o-Chrom column (300 A pore diameter packed with Reliasil
C,,, 2 mm X 10 cm, Upchurch Scientific, WA, USA) using an ABI 140B dual pump
solvent delivery system (Applied Biosystems, Foster City, CA, USA) pumping at a
flowrate of 75 uL/min. Peek materials were utilized instead of stainless steel to minimize
99
sample losses due to interaction of phosphopeptides with iron. Peptides were separated by
reverse phase high performance liquid chromatography (HPLC) and eluted utilizing a 60
min gradient of 0-80% Solvent B (70:30:0.085 acetonitrile (HPLC grade, EM Science,
Gibbstown, NJ, USA):water:TFA, v:v:v). Peptides were detected by monitoring UV
absorbance at a wavelength of 220 nm. Fractions were collected into polypropylene
microcentrifuge tubes, lyophilized to dryness (Savant Instruments, Farmingdale, NY,
USA) and stored at -4°C. Samples were subsequently reconstituted in 10 - 15 WL of 0.1%
TFA prior to analysis.
3.3.3.2.2_ Edman Degradation
Manual Edman degradation was performed by reconstituting lyophilized fractions
- in 10 WL of 5% phenylisothiocyanate in pyridine added to 10 LL of 50% aqueous pyridine
followed by heating at 37°C for 30 min. The organic layer was extracted twice with 20 WL
of 2:1 heptane:ethyl acetate and the aqueous material lyophilized. Cleavage of the N-
terminal amino acid residue was accomplished by adding 10 uL of TFA to the sample,
heating at 37°C for 15 min, then lyophilizing. A final extraction was made using 30 uL of
water combined with 50 WL of n-butyl chloride. The sample was lyophilized then
reconstituted in 0.1% TFA and applied to the probe tip as described below.
3.3.3.2.3 MALDI
Matrix consisted of a saturated solution of o-cyano-4-hydroxycinnamic acid
(Aldrich Chemical Co., Milwaukee, WI, USA) in an equivolume mixture of 0.1% TFA
and acetonitrile. A 1/2 WL aliquot of sample was co-deposited onto a gold-plated stainless
100
steel probe tip with 1 uL of matrix and allowed to air dry. The probe was then inserted
into the vacuum chamber utilizing a ball valve and the laser was employed to ionize the
sample.
3.3.3.3, Data Analysis
A database consisting of the transcribed genetic sequence of the P protein was
constructed. The PEPM™ (Finnigan MAT) algorithm (51) was used to identify all
possible sequences of amino acid residues in the database with molecular weights within
+/- 5 u of the experimentally determined molecular weight for the peptide of interest. The
software also listed the predicted y-, b- and a-type ions (nomenclature described in (52)) to
aid in the. analysis of the fragmentation mass spectra. SEQUEST, a database searching
algorithm developed in our laboratory (53), was employed to confirm the identification of
the amino acid sequences. A list of possible sequences in the P protein database was
generated and theoretical fragmentation mass spectra constructed. The experimental
fragmentation mass spectra were then compared to theoretical fragmentation mass spectra,
scored, and ranked. Peaks in the mass spectra labeled with a "P" refer to fragmentation
products generated from a phosphorylated peptide. The notation "y" refers to the loss of a
| guanidino group from an Arg-containing ion while "*" refers to the loss of water or
ammonia. Peaks labeled with b,y,, refer to internal cleavage products from fragmentation
at Pro, His, or Arg residues, e.g., given a peptide sequence LPQGW, b,y, corresponds to
PQGW, b.,y, corresponds to PQG, etc.
101
3.3.4 Results and Discussion
3.3.4.1 Peptide Mapping
Purified Sendai virus P protein was enzymatically digested using trypsin. Resultant
peptides were separated by HPLC, fractions collected, and samples for mass spectrometric
analysis prepared as described above. The MALDI ion trap and the MALDI TOF were
used to screen the fractions for phosphopeptides and to provide a peptide map of the P
protein. The ion trap was employed to obtain molecular weight information. The TOF
was used to determine the number of peptides in each fraction since the ion trap mass
spectra were complicated by the presence of fragmentation products. Some degree of
fragmentation of peptide ions upon injection into ion trap ‘mass spectrometers is common.
The decomposition provided by metastable decay using the matrix o-cyano-4-
hydroxycinnamic acid affords abundant sequence-specific fragmentation products which
can be diagnostic for the structure of known biological molecules (54-58). The sequence-
specific fragmentation produced upon injection into the ion trap provided verification of
peptide assignment without the need to perform an additional tandem mass spectrometry
experiment. Figure 3.9 shows the amino acid sequence of the P protein and the sequence ~
coverage obtained by tryptic mapping. The tryptic map covered ~61% of the protein by
mass with most of the coverage on the N-terminal half of the protein where the
phosphorylation sites were expected (39, 45). Trypsin digestion produces 71 expected
peptides. Thirty-four peptides were mapped, representing 48% of the expected trypsin
fragments. The molecular weights for 25 of the peptides not identified were below the
low-mass cutoff determined by the matrix ejection pulse and were not detected. The
remaining 12 peptides accounted for 26% of the sequence and were not unambiguously
102
Figure 3.9: Amino acid sequence of the P protein of Sendai virus. Underlined residues
are from peptides identified by tryptic mapping. The letter “c’” below a residue indicates
the phosphopeptide identified from the chymotrypsin digest, the letter “t” below a residue
indicates the phosphopeptide identified from the trypsin digest, and the letter "o" indicates
residues from a peptide overlapping that identified from the chymotrypsin digest.
103
MDODAFILKE DSEVEREAPG GRESLSDVIG FLDAVLSSEP TDIGGDRSWL
’ HNTINTPOGP
GSAHRAKSEG
EGEVSTPSTO DNRSGEESRV SGRTISKPEAE
AHAGNLDKON
TEDENREMAA
IHRAFGGRTIG _TNSVSODLGD GGDSGILENP PNERGYPRSG
HPDKRGEDOA
EGLPEEVRGS TSLPDEGEGG ASNNGRSMEP
GSSHSARVTG
PLNRYNSTGS
VLVIPSPELE
PPGKPPSTOD
EAVLRRNKRR_PTNSGSKPLT. PATVPGTRSP
Cc cecececoece
EHINSGDTPA VRVKDRKPPI_ GTRSVSDCPA
cc tttttt
NGRSIHPGLE
SYVFARRALK
cttttttttt
TDSTKKGIGE
SANYAEMTFN
tetttttttt tt
NTSSMKEMAT LLTSLGVIOS AOQEFESSRDA
VCGLILSAEK SSARKVDENK OLLKOTOESV
ESFRDTYKRF SEYOKEONSL LMSNLSTLHI_ ITDRGGKTDN TDSLTRSPSV
FAK SKENKTK ATRFDPSMET LEDMKYKPDL IREDEFRDEI RNPVYQERDT
EPRASNASRL LPSKEKPTMH SLRLVIESSP LSRAEKAAYV KSLSKCKTDO
EVKAVMELVE
EDIESLTN
104
identified. The P protein was also digested with chymotrypsin to identify phosphopeptides
not observed in the trypsin digest.
3.3.4.2 Trypsin Digestion
3.3.4.2.1 MALDI/Time-of-Flight Mass Spectrometry
One trypsin-generated peptide from HPLC fraction #16 with m/z 2911 was
detected. The mass spectrum illustrated in Figure 3.10 shows detection of two major
peaks with m/z values of 2911 and 2991. These differ in mass by 80 u, corresponding to
the addition of HPO, to the hydroxyl group on the side chain of Ser. The mass difference
suggests that the lighter peptide is the non-phosphorylated version of the heavier peptide.
The mass corresponds to that of the tryptic peptide 255-282 and confirmation of the
assignment using the ion trap mass spectrometer is discussed below. Four serine residues
are contained within the sequence, thus the site of phosphorylation cannot be assigned
based on the molecular weight data.
3.3.4.2.2_ MALDI/Ion Trap Mass Spectrometry
Analysis of trypsin-generated HPLC fraction #16 afforded the mass spectrum
shown in Figure 3.11. The dominant peak in the spectrum was at m/z 2909, correlating
with the signal observed using the TOF mass spectrometer. A small peak at m/z 2989 was
observed. The mass difference of 80 u again indicates that the heavier peptide was
probably the phosphorylated analog of the lighter peptide. A strong signal is also observed
at m/z 2890. This signal corresponds both to a loss of ~98 u (neutral loss of phosphoric
acid from the phosphopeptide at m/z 2989) and a loss of water from the unphosphorylated
105
Figure 3.10: Linear time-of-flight mass spectrum for the phosphopeptide of m/z 2909
resulting from trypsin digestion of the P protein. Ten laser shots were summed.
Relative Abundance
106
~ (M+H+HPO3)*
2991
(M+H)t
2911
2400
2600
2800 3000 3200 3400 3600
m/z
107
Figure 3.11: Fragmentation mass spectrum for the phosphopeptide of m/z 2909 resulting
from trypsin digestion of the P protein. The ion trap was set to stably trap ions with m/z
values larger than 90 u. Matrix ions were ejected by ramping the amplitude of the rf
voltage from 20 u to 650 u while applying a supplementary signal at 520,300 Hz, 5.6 V
(peak-to-peak, endcap-to-endcap). The mass spectrum was acquired by ramping the
amplitude of the rf voltage from 35 u to 650 u while applying a supplementary signal at
59,124 Hz, 8.0 V (peak-to-peak, endcap-to-endcap). (a) Sequence and expected m/z values
of fragmentation products for the unphosphorylated form of residues 255-282. Observed
ions are underlined. Ions corresponding to phosphorylated fragmentation products are
shown above (b-type ions) and below (y-type ions) the unphosphorylated fragmentation
products. (b) Fragmentation mass spectrum for m/z 2909. (c) Result of SEQUEST
analysis.
108
6ES}
ufD
OOS
GLL_ ble Sve e2@rv ErS 859 SIZ c08 916 6c0l ZOLL 96cl IDL
ZIV A UV Od WE dsy AID 2S usy AT SHH MID dsy
lose Sele 9692 s9Se BONE ZOEC cGec Sle 8Ole v66l 1eBl PYLE SIOL
LL6¢ 91Ze Lbye cee Slee vL0c S691
Zove ZiZe 9e82 Be6e
Z2Z. pZ8t Te6l 6pOe 9Ole 02d NNEC Lees Pree GSE cE9C YHLE 60Ee
DS Old O1g SAT AID Old O1G ASG AID AL WS usy AA],
LZZL v8tt 9801 686 198 vos ZOL O19 E2S 99h SG9E B42 VIL
6901
b)
He
TP WNE |
Relative Abundance
2000 2200
Rank/Sp (M+H)+ cn
1/l 2911.1 1.0000 77.
2/3 2910.2 0.3621 28.
3/8 2908.2 0.3353 15.
4/6 2910.1 0.2894 17
5/4 2911.2 0 22
1007
807
1007
807]
607
407
207.4
800
2225
109
DinYg
1000
1200
Yi2
1400 1600 1800
(M+H)t
_)
”S (M+H+HPO3)t
2400 2600 2800 3000
m/z
Ions Peptide
14/81 (R) YNSTGSPPGKPPSTQDEHINSGDTPAVR
8/81 (A) TVPGTRSPPLNRYNSTGSPPGKPPSTQD
6/84 (S) GSKPLTPATVPGTRSPPLNRYNSTGSPPG
7/75 (-) MDOQDAFP ILKEDSEVEREAPGGRESLS
7/66 (L) LKQIQESVESFRDTYKRFSEYOK
110
peptide. This loss of 98 has been shown to be a signature for the presence of
phosphorylated serine and threonine (58-60).
A search of the P protein sequence using PEPM™ identified 37 possible peptides
with m/z values within +/-5 u.of 2909. Of these, five contained Lys or Arg at the C-
terminus, corresponding to peptides produced by trypsin digestion. Only one of the five
sequences corresponded to an expected tryptic fragment, residues 255-282. Theoretical
values of b- and y-type ions for the sequence were calculated and compared to the fragment
ions observed in the mass spectrum, shown in Figure 3.11(b). The observed fragment
ions correspond to the product ions expected for the tryptic fragment 255-282,
YNSTGSPPGKPPSTQDEHINSGDTPAVR, confirming the sequence assignment made
using TOF analysis. The expected fragmentation products for the unphosphorylated
sequence are displayed in Figure 3.11(a) and observed ions underlined. Unphosphorylated
sequence ions corresponding to the major peaks in the mass spectrum were identified as
were minor peaks arising from fragmentation of the phosphorylated species. The presence
of a series of y- and b-type ions served to confirm the sequence assignment. Signal
suppression of b-type ions is due to the large number of proline residues present. A
number of signals were present corresponding to internal cleavages of the peptide
modulated by proline and histidine residues. These peptides provided particularly strong
signals when the C-terminal amino acid was Asp, in agreement with recent observations
(58). The signal from the phosphorylated molecular ion was weak and a series of low
abundance peaks afforded by fragmentation of the phosphopeptide were present. The
presence of the y,, ion as well as its phosphorylated analog indicate that Ser-260 is the
most likely site of phosphorylation. The phosphorylated analogs Of Yo... aS well as by,
bias Digs Dios Do1.732 Dog and bz, also serve to confirm the identification. Contributions to low
111
abundance signals can also arise from the presence of other co-eluting peptides. Site-
directed mutagenesis experiments (47) have shown that - S P - is a consensus sequence for
phosphorylation of the P protein. The phosphopeptide generated using the trypsin digest ©
contains four serine residues at Ser-257, Ser-260, Ser-267, and Ser-275.. Only Ser-260 is
followed by a proline; thus, the identification of Ser-260 as the phosphorylated residue
would agree with the proposed mechanism of a proline-mediated kinase.
The SEQUEST database searching program was subsequently used to confirm
the sequence assignment. Results in Figure 3.11(c) indicate that the expected sequence was
chosen as the first-ranked choice. The final ranking is determined by the value for C, and
~ the answer is assumed to be reliable if the C,, value for the first choice is much greater than
the C, value for the second choice (53). This is the case for the sequence assignment in
Figure 3.11(c); thus, the manually determined answer was verified by computer-aided
interpretation. Additional stages of mass spectrometry were done on the most abundant
fragmentation products (MS/MS and MS’); however, no additional sequence information
was obtained. Sub-digestion of the fraction on the probe tip using Asp-N was attempted,
but the resulting peptides were not detected. The same phosphorylated peptide sequence
was identified using sample extracted from the 2D-TLE experiment (data not shown).
The SEQUEST program is capable of searching sequences for post-translational
modifications, such as phosphorylation. For the phosphopeptides identified in this work,
the best results were obtained by searching using the unphosphorylated mass on a
sequence database where phosphorylation was not included. We infer from this that the
facile loss of phosphoric acid from the phosphopeptide tends to suppress further
fragmentation; therefore, fragmentation proceeds primarily from the unphosphorylated
species. Similar effects have been observed on another MALDI-ITMS (57).
112
3.3.4.3 Chymotrypsin Digestion
3.3.4.3.1 MALDI/Time-of-Flight Mass Spectrometry, Fraction #13
In previous work (54), digestion with chymotrypsin and subsequent 2D-TLE of
the peptides produced two spots. In the present work, two candidate phosphopeptides
were also observed when screening the fractions collected from the chymotrypsin digest.
Two major peaks of m/z 1738 and m/z 1807 from HPLC fraction #13 were detected in the
mass spectrum displayed in Figure 3.12. The 69 u difference between the peaks does not
correlate well with the expected 80 u mass difference between ions resulting from the
phosphorylated and unphosphorylated forms of the peptide. However, subsequent
analysis of this fraction by ion trap mass spectrometry revealed the presence of a co-eluting
peak at m/z 1728, corresponding to the non-phosphorylated version of the peptide of m/z
1807. The broadened peak in Figure 3.12 that was assigned to m/z 1738 results from the
combined presence of m/z 1728 and m/z 1743, a co-eluting peptide identified as the
chymotryptic fragment HIITDRGGKTDNTDSL, residues 429-444. The low resolution
linear TOF mass spectrometer was not able to discriminate between the peaks at that
molecular weight. The peptide of m/z 1728 corresponds to the chymotryptic fragment
from residues 240-255. Ser-249 is the only serine residue contained in this sequence; thus
the site of phosphorylation was straightforward to assign.
3.3.4.3.2, MALDI/Ion Trap Mass Spectrometry, Fraction #13 .
Analysis of chymotrypsin-generated HPLC fraction #13 produced the mass
spectrum shown in Figure 3.13(b). The dominant peak in the mass spectrum was at m/z
1709. An abundant signal at m/z 1807 correlated with that observed on the time-of-flight
113
Figure 3.12: Linear time-of-flight mass spectrum for the phosphopeptide of m/z 1728
resulting from chymotrypsin digestion of the P protein. Ten laser shots were summed.
The mass assignment for the (M+H)* peak is incorrect due to the presence of a co-eluting
peptide that served to broaden the peak. The mass resolution of the instrument was too
low to separate the peaks.
Relative Abundance
26 7
25 |
24 |
23 7
22 |
21 5
20 °
19 |
18
17 7
114
(M+H+HPO3)*
1807
(M+H)t
1738
1200
1400
1600 1800
m/z
2000
2200
2400
2600
115
instrument. A small peak at m/z 1728 was also observed. The mass difference of 79 u
indicates that the peptide at m/z 1728 is the non-phosphorylated version of the peptide at
m/z 1807. The mass difference of 98 u between the peaks at m/z 1807 and m/z 1709
suggests that the lighter peptide results from the facile loss of phosphoric acid from the
heavier peptide as well as from the loss of water from m/z 1728. The presence of a low
abundance signal from a co-eluting peak at m/z 1743 corresponding to residues 429-444
was also observed.
The PEPM™ software was used to search the P protein database for amino acid
sequences with the mass 1728 Da. Of 36 possible peptides identified, 7 contained a
C-terminal residue resulting from chymotrypsin cleavage on the carboxyl side of tyrosine,
tryptophan, leucine, or phenylalanine. Five of these peptides corresponded to expected
chymotrypsin-generated fragments. One step of manual Edman degradation was
performed as described above, and the resulting mass spectrum is depicted in Figure 3.14.
The mass-to-charge ratios of the ions shifted by approximately 100 u, e.g., 950 u — 850
u, 1487 u — 1388 u, 1667 u > 1567 u, 1709 u > 1610 u, indicating valine or threonine
as likely candidates for the N-terminal residue. Only two peptides out of the five
possibilities contained Val or Thr at the N-terminus. Expected values for b- and y-type
ions were calculated and compared with the experimental spectrum in Figure 3.13(b). The
sequence of the chymotryptic peptide TPATVPGTRSPPLNRY, residues 240-255, fit the
experimental data, strongly suggesting the phosphorylation site was at Ser-249. This
peptide was also generated as the top-ranked choice by SEQUEST, shown in Figure
3.13(c). The expected fragmentation products for the peptide sequence are displayed in
Figure 3.13(a) and observed ions are underlined. The presence of a series of b- and y-type
ions served to confirm the sequence identification. Some signal suppression following Pro
116
Figure 3.13: Fragmentation mass spectrum for the phosphopeptide of m/z 1728 resulting
from chymotrypsin digestion of the P protein. The ion trap was set to stably trap ions with
__ m/z values larger than 100 u. Matrix ions were ejected by ramping the amplitude of the rf
voltage from 20 u to 650 u while applying a supplementary signal at 520,300 Hz, 5.6 V
(peak-to-peak, endcap-to-endcap). The mass spectrum was acquired by ramping the
amplitude of the rf voltage from 30 u to 650 u while applying a supplementary signal at
89,202 Hz, 10.4 V (peak-to-peak, endcap-to-endcap). (a) Sequence and expected m/z
values of fragmentation products for the unphosphorylated form of residues 240-255.
Observed ions are underlined. ons corresponding to phosphorylated fragmentation
products are shown above (b-type ions) and below (y-type ions) the unphosphorylated
fragmentation products. (b) Fragmentation mass spectrum for m/z 1728. (c) Result of
SEQUEST analysis.
117
cet sot
UAL, 31V
o8ht
ZSp G9S 299 6&2
usSy Noe] Old Old
60Z- 9OPSl
B21
O6E+ QZcl E9ll QOL
OLvl crcl
cO00l
31V
+88
Wer OT.
POLL TOLLE SSCL ZSEL SSrl 6BcSl
WL AID old TA AL eV
Gel ve9 L295 OLb Ile OL?
ZO8t
9c9L LcoZLk
o1g IL
661 ZOl
(e
b)
Pr.
B® WN
Relative Abundance
100)
80-
100;
804
607
1300
Y6
1400
ooo COF
1500
Cn
.0000
-5704
-4490
.3178
.7774
118
1600
m/z
1100. +1200
big
/ +
* +
omy
1700 1800
Ions Peptide
9/45 (L) TPATVPGTRSPPLNRY
4/45 (G) GKTDNTDSLTRSPSVF
7/45 (L) RLVIESSPLSRAEKAA
6/42 (E) SSRDASYVFARRALK
2/45
(P) STODEHINSGDTPAVR
119
or Gly was observed. A loss of 43 u, corresponding to the loss of the guanidino group
from b-type ions containing arginine, was observed for most of the b-ions. Similar losses
have been observed for the Arg-containing model peptide angiotensin using this
instrument. The presence of phosphorylated analogs of b,, and y,, as well as Ys, Yip, Dy,
b,,, and b,, afforded positive identification of Ser-249 as the site of phosphorylation. The
Edman degradation results (Figure 3.14) confirm the identification as well with the
presence of the phosphorylated analogs of b, and y,, as well as b,,,b,,, and y,,. An internal
cleavage product RSPPLN, as well as its phosphorylated analog, were also observed.
Further confirmation for the sequence identification was provided by analysis using
electrospray ionization on a TSQ700 triple quadrupole mass spectrometer (data not shown)
and by site-directed mutagenesis. The identification of the site of phosphorylation agrees
with the proline-mediated kinase mechanism discussed above (47). The same results were
obtained by analysis of peptides extracted from one of the spots on the 2D-TLE gel (data
not shown).
3.3.4.3.3 MALDI/Time-of-Flight Mass Spectrometry, Fraction #25
The mass spectrum depicted in Figure 3.15 results from analysis of HPLC fraction
#25 and shows the presence of two major peaks. The signals at m/z 2732 and m/z 2811
differ by 79 u; thus the lighter peptide appears to be the non-phosphorylated version of the
heavier peptide. The mass of this peptide did not correspond to an expected chymotryptic
fragment. Further analysis by ion trap mass spectrometry (see below) was used to assign
the peptide to residues 228-253, a peptide overlapping that identified from HPLC fraction
#13.
120
Figure 3.14; Fragmentation mass spectrum resulting from one stage of manual Edman
degradation of m/z 1728. Operating conditions were as described for the analysis of m/z
1728.
121
.COdH+H+W)
(M+H+HPO, - 99.5)*
(M+H+HPO, - H,PO, - 99.5)*
4($'66 - H+) —_,
100;
80 4
XO +
QouRpUNGyY dajeyay
1800
1600
an)
\O
m/z
122
3.3.4.3.4 MALDI/Ion Trap Mass Spectrometry, Fraction #25
Mass spectrometric analysis of chymotrypsin-generated HPLC fraction #25
afforded the mass spectrum depicted in Figure 3.16 (b). The dominant peak in the
spectrum was at m/z 2710 and an abundant signal was also observed at m/z 2728,
correlating with data obtained by TOF methods. The phosphorylated analog appeared with
low abundance at m/z 2809, an 80 u mass difference from the peak at m/z 2728 and a 98 u
mass difference from the peak at m/z 2710. Again, a facile loss of phosphoric acid was
observed.
PEPM™ was used to search the database for all peptides with m/z values of 2730 u
+/-5 u. The search produced 35 possible peptides. Of these, four sequences theoretically
resulted from cleavage with chymotrypsin. Signals at 2616 u and 2599 u were used to
generate possibilities for the largest b- and y-type ions. The largest y-type ion could
correspond to a loss of 112 u or 129 u, indicating L, I, N, K, Q, and E as possibilities. The
largest b-type ion could correspond to a loss of 95 u or 112 u, indicating L, I, N, and P as
possibilities. Sequences for all peptides with these possible terminating ions were
investigated. Mass assignment errors in the ion trap can arise from the use of a two-point
calibration rather than a five-point calibration for the extended mass range, as well as
shifting caused by space-charge effects; thus the mass windows considered were rather
large. An MS/MS experiment (data not shown) indicated that the peaks at m/z 2175 and
1294 were from the precursor ion at m/z 2730. Expected sequence ions for 20 possible
sequences were compared with the experimental mass spectrum. The sequence with the
best fit corresponded to residues 228-253,
123
Figure 3.15: Linear time-of-flight mass spectrum for the phosphopeptide of m/z 2730
resulting from chymotrypsin digestion of the P protein. Ten laser shots were summed.
Relative Abundance
124
(M+H+HPO3)t
(M+H)* 2732 2811
5.6
5.24
4.8)
44:
4.07
3.65
3.2!
" 2300 2500 2700 | 2900 | 3100
m/z
1700 +1900 ~—-2100
125
Figure 3.16: (a) Sequence and expected m/z values of fragmentation products for the
unphosphorylated form of residues 228-253. Observed ions are underlined. Jons
corresponding to phosphorylated fragmentation products are shown above (b-type ions)
and below (y-type ions) the unphosphorylated fragmentation products. (b) Fragmentation
mass spectrum for phosphopeptide of m/z 2730 resulting from chymotrypsin digestion of
the P protein. The ion trap was set to stably trap ions with m/z values larger than 120 u.
Matrix ions were ejected by ramping the amplitude of the rf voltage from 20 u to 500 u
while applying a supplementary signal at 242,300 Hz, 9.6 V (peak-to-peak, endcap-to-
endcap). The mass spectrum was acquired by ramping the amplitude of the rf voltage
from 50 u to 650 u while applying a supplementary signal at 35,371 Hz, 5.6 V (peak-to-
peak, endcap-to-endcap).
126
bd 8B BriT
k €&l Ore eve Ory LEG €89 Pp8L IPS 8E6 SEO SELL
usy Noy O1g Og Sq BAY AYL AT) Old [eA IY
q TtZe Z6S¢c rere LBEC OB20 bO0Cc LH0d QV6l 688l CBZ C6OL
Od L9¢6
sal
O6cl ZB8El LOOL 8691 clot Z90¢ ILle cloc L89¢
OlzlL ZOGl BOrpr FoSl SLOT OPZt ECB O68l LZ6l 1602 c6le 68ce She 1096 bale
UV Old IL nay o1g SAY wg AID JS Usy AL 1d diy s1y shy
c6St beSt vebl €ZElL OlZL ELLE 86 Z68 Ors ESL 6E9 BES Ih GBC Gel
(e
127
8KF&q o H+)
Q1 fern =O : =
Ilq~~.. oN Wat
sf!
+m;
ved ! i
“N Ss
s Qu
a) . :
= 7 CeK_g ---
S we
Ih-g
Ex¥q- --
S q-d---
Erg.
S oo-*A"a-d
rc
Lktq ~~.
i)
i)
oo
S I K-g aK i ihe
= * S a ‘© t
aol ‘
ee
= dUBPUNGY daAljelay
128
KRRPTNSGSKPLTPATVPGTRSPPLN. This peptide overlaps the phosphopeptide with
m/z 1728 from residues 240-255. The expected fragmentation products appear in Figure
3.16(a) and observed signals are underlined. The presence of a series of b- and y-type ions
were sufficient to verify the sequence assignment. As expected, fragmentation was
suppressed in the vicinity of proline residues and signal from a number of internal cleavage
products was observed. There are 3 serine residues found in this peptide. Since only peak
differences of 80 u were observed using the TOF, we assume that the overlap peptide was
phosphorylated at only one site. The observation of phosphorylated analogs of b,;, Y.5, Yoo.
o> Yis> Yie1s° Yisix Yio» and y, serve to confirm the site of phosphorylation on Ser-249
rather than on Ser-234 or Ser-236. In addition, only Ser-249 is followed by a proline
residue, agreeing with the proposed kinase mechanism (47). The same results were
obtained by analysis of peptides extracted from one of the spots on the 2D-TLE gel (data
not shown).
3.3.5 Conclusion
Three phosphopeptides were observed using MALDI/quadrupole ion trap mass
spectrometry. Digestion with trypsin produced one phosphopeptide corresponding to
residues 255-282, while digestion with chymotrypsin produced two phosphopeptides that
overlapped in sequence. The smaller peptide corresponding to residues 240-255 contained
one serine residue at Ser-249. The larger overlap peptide corresponded to residues 228-
253 with Ser-249 again identified as the site of phosphorylation.
129
In parallel work using site-directed mutagenesis (47), deletion mutants were
screened to locate a putative region of phosphorylation. The dominant phosphorylation
region corresponded to residues 238-254 containing one serine at Ser-249. Deletion of
Ser-249 or mutagenesis in that region of the peptide resulted in more extensive
phosphorylation of the minor phosphopeptides, particularly a minor phosphopeptide
located between residues 253 and 316. Experiments to investigate the effect of deleting
residues 253 - 316 on the overall state of phosphorylation were not carried out. Concurring
with these results, we believe Ser-249 is indeed a dominant phosphorylated residue in the P
protein. The question remains as to why the phosphorylated peptide fragment containing
Ser-260 was observed using mass spectrometry and not detected, except as a minor
species, by classical genetics experiments. One possibility is that the fragment ionized
particularly well by MALDI and that the other minor phosphopeptides were not observed
because of low stoichiometries. Another possibility is that the structure of the region of the
protein surrounding Ser-260 precluded efficient incorporation of labeling agents, reducing
its detectability by autoradiography. More work is required to determine the exact location
of the phosphorylation site of the peptide fragment.
The functional significance of P protein phosphorylation is | unclear.
Phosphorylation has been proposed to aid in multimerization of the P protein and the
maintainance of its structural integrity (47), enabling viral replication and transcription.
With the determination of the phosphorylation sites on the P protein, classical genetics can
be employed to elucidate the precise role of phosphorylation in Sendai virus functioning.
130
3.4 References
1. Karas, M., Bachmann, D., Bahr, U., and Hillenkamp, F. (1987) Int. J. Mass
Spectrom. Ion Proc. 78 53-68.
2. Tanaka, K., Waki, H., Ido, Y., Akita, S., and Yoshida, Y. (1988) Rapid Commun.
Mass Spectrom. 2 151-153.
3, Karas, M., Bahr, U., Ingendoh, A., Nordhoff, E., Stahl, B., Strupat, K., and
Hillenkamp, F. (1990) Anal. Chim. Acta 241 175-185.
“A, ‘Hillenkamp, F., Karas, M., Beavis, R. C., and Chait, B. T. (1991) Anal. Chem. 63
1193A-1202A.
5. Beavis, R. C., and Chait, B. T. (1990) Proc. Natl. Acad. Sci. USA 87 6873-6877.
6. - Hill, J. A., Annan, R. S., and Biemann, K. (1991) Rapid Commun. Mass
Spectrom. 5 395-399.
7. Annan, R. S., Kochling, H. J., Hill, J. A., and Biemann, K. (1992) Rapid
Commun. Mass Spectrom. 6 298-302.
8. Hettich, R. L., and Buchanan, M. V. (1991) J. Am. Soc. Mass Spectrom. 222-28.
9. Hettich, R. L., and Buchanan, M. V. (1991) J. Am. Soc. Mass Spectrom. 2 402-
412.
10. Castro, J. A., Koster, C., and Wilkins, C. (1992) Rapid Commun. Mass Spectrom.
6 239-241.
11. Cox, K. A., Williams, J. D., Cooks, R. G., and Kaiser, R. E. (1992) Biol. Mass
Spectrom. 21 226-241.
12. Russell, D. H., and Solouki, T. (1992) Proc. Natl. Acad. Sci. USA 89 5701-5704.
131
13. Chambers, D., Goeringer, D. E., McLuckey, S. A., and Glish, G. (1993) Anal.
Chem. 65 14-20.
14. _—‘ Kaiser, R. E., Jr., Cooks, R. G., Syka, J. E. P., and Stafford, G. C., Jr. (1990)
Rapid Commun. Mass Spectrom. 430-33. |
15. Hunt, D. F., Shabanowitz, J., Yates, J. R., I, Zhu, N.-Z., Russell, D. H., and
Castro, M. (1987) Proc. Natl. Acad. Sci. USA 84 620-623. |
16. Cody, R. B., Jr., Amster, I. J., and McLafferty, F. W. (1985) Proc. Natl. Acad. Sci.
USA 82 6367-6370.
17. _ Beavis, R. C., and Chait, B. T. (1991) Chem. Phys. Lett. 181 479-484.
18. Louris, J. N., Amy, J. W., Ridley, T. Y., and Cooks, R. G. (1989) Int. J. Mass
Spectrom. Ion Proc. 88 97-111.
19. Louris, J. N., Brodbelt-Lustig, J. S., Kaiser, R. E., Jr., and Cooks, R. G. (1988)
Proc. of the 36th ASMS Conf. Mass Spectrom. and Allied Topics, June 5-10, San
Francisco, CA, American Society for Mass Spectrometry, p. 968. |
20. Heller, D. N., Lys, I., Cotter, R. J., and Uy, O. M. (1989) Anal. Chem. 61 1083-
1086.
21. Glish, G. L., Goeringer, D. E., Asano, K. G., and McLuckey, S. A. (1989) Int. J.
Mass Spectrom. Ion Proc. 94 15-24.
22. Goeringer, D. E., Glish, G. L., and McLuckey, S. A. (1991) Anal. Chem. 63
1186-1192.
23. Jonscher, K., Currie, G., McCormack, A. L., and Yates, J. R., II (1992) Proc. of
the 40th ASMS Conf. on Mass Spectrom. and Allied Topics, May 31-June 5, Washington,
DC, American Society for Mass Spectrometry, p. 701.
132
24. Bier, M. E., Schwartz, J., Jardine, I., and Stafford, G. C. (1992) Proc. of the 40th
ASMS Conf. on Mass Spectrom. and Allied Topics, May 31-June 5, Washington, DC,
American Society for Mass Spectrometry, p. 1017.
25. Kaiser, R. E., Jr., Louris, J. N., Amy, J. W., and Cooks, R. G. (1989) Rapid
Commun. Mass Spectrom. 3 225-229. |
26. Louris, J. N., Cooks, R. G., Syka, J. E. P. Kelley, P. E., Stafford, G. C., Jr., and
Todd, J. F. J. (1987) Anal. Chem. 59 1677-1685.
27. Kaiser, R. E. (1990), Ph.D. Dissertation, Purdue University.
28. Wright, L. G., Cooks, R. G., and Wood, K. V. (1985) Biomed. Mass Spec. 12
159-162.
29. Schwartz, J. C., Syka, J. E. P., and Jardine, I. (1991) J. Am. Soc. Mass Spectrom.
2 198-204.
30. Beavis, R. C., and Chait, B. T. (1989) Rapid Commun. Mass Spectrom. 3 233-
237. | |
31. Beavis, R. C., and Chait, B. T. (1989) Rapid Commun. Mass Spectrom. 3 432-
435. |
32. Doktycz, S. J., Savickas, P. J., and Kreuger, D. A. (1991) Rapid Commun. Mass
Spectrom. 5 145-148.
33. Kingsbury, D. W. (1991) in Fundamentals of Virology (Fields, B. N., and Knipe,
D. M., Eds.), Raven Press, New York, pp. 507-524.
34. Dillon, P. J., and Gupta, K. C. (1989) J. Virology 63 974-977.
35. Vidal, S., Curran, J., and Kolakofsky, D. (1990) J. Virology 64 239-246.
36. Hamaguchi, M., Yoshida, T., Nishikawa, K., Naruse, H., and Nagai, Y. (1983)
Virology 128 105-117.
133
37. Horikami, S. M., Curran, J., Kolakofsky, D., and Moyer, S. A. (1992) J. Virology
66 4901-4908.
38. Lamb, R. A., Mahy, B. W. J., and Choppin, P. W. (1976) Virology 69 116-131.
39. Hsu, C., and Kingsbury, D. W. (1982) Virology 120 225-234.
40. | Curran, J., and Kolakofsky, D. (1990) Enzyme 44 244-249.
41. | Curran, J., Boeck, R., and Kolakofsky, D. (1991) EMBO J. 10 3079-3085.
42. Matsuoka, Y., Curran, J., Pelet, T., Kolakofsky, D., Ray, R., and Compans, R. W.
(1991) J. Virology 65 3406-3410.
43. Smallwood, S., Ryan, K. W., and Moyer, S. A. (1994) Virology 202 154-163.
44. Curran, J., Pelet, T., and Kolakofsky, D. (1994) Virology 202 875-884.
45. Vidal, S., Curran, J., Orvell, C., and Kolakofsky, D. (1988) J. Virology 62 2200-
2203. |
46. Byrappa, S., Hendricks, D. D., Pan, Y.-B., Seyer, J. M., and Gupta, K- C. (1995)
. Virology 208 408-413.
47. Byrappa, S., Pan, Y.-B., and Gupta, K. C. (1996) Virology 216 228-234.
48. Jonscher, K. R., Currie, G., McCormack, A. L., and Yates, J. R., III (1993) Rapid
Commun. Mass Spectrom. 7 20-26.
49. Morand, K. L., Cox, K. A., and Cooks, R. G. (1992) Rapid Commun. Mass
Spectrom. 6 520-523.
50. Kaiser, R. E., Jr., Cooks, R. G., Stafford, G. C., Jr., Syka, J. E. P., and
Hemberger, P. H. (1991) Int. J. Mass Spectrom. Ion Proc. 106 79-115.
51. Watkins, P. J. F., Jardine, ., and Zhou, J. X. G. (1991) Bioch. Soc. Trans. 19
957-962.
134
52. Roepstorff, P., and Fohlman, J. (1984) Biomed. Mass Spectrom. 11 601.
53. Eng, J. K., McCormack, A. L., and Yates, J. R., II (1994) J. Am. Soc. Mass
Spectrom. 5 976-989.
54. Jonscher, K. R., and Yates, J. R., III (1994) Proc. of the 42nd ASMS Conf. on
Mass Spectrom. and Allied Topics, May 29-June 3, Chicago, IL, American Society for
Mass Spectrometry, pp. 216-217.
55. Qin, J., and Chait, B. T. (1995) Proc. of the 43rd ASMS Conf. on Mass Spectrom.
and Allied Topics, May 21-26, Atlanta, GA, American Society for Mass Spectrometry, p.
1100.
56. Qin, J., and Chait, B. T. (1995) J. Am. Chem. Soc. 117 5411-5412.
57. Qin, J., and Chait, B. T. (1995) Proc. of the 43rd ASMS Conf. on Mass Spectrom.
and Allied Topics, May 21-26, Atlanta, GA, American Society for Mass Spectrometry, p.
989. |
58. Qin, J., Steenvoorden, R. J. J. M., and Chait, B. T. (1996) Anal. Chem. 68 1784-
1791.
59. Gibson, B. W., and Cohen, P. (1990) Meth. Enzym. 193 480-501.
60. Jonscher, K. R., and Yates, J. R., III (1993) Proc. of the 41st ASMS Conf. on Mass
Spectrom. and Allied Topics, May 31-June 4, San Francisco, CA, American Society for
Mass Spectrometry, 695a.
135
Chapter 4
Mixture Analysis Using a Quadrupole Mass Filter/Quadrupole
Ion Trap Mass Spectrometer
4.1 Overview
A hybrid tandem mass spectrometer is constructed by interfacing a quadrupole
mass filter (Q) to a quadrupole ion trap mass spectrometer (QITMS) and is evaluated for
the analysis of mixtures. The mass filter is set to selectively inject ions of a particular m/z
or, in scanning mode, to sequentially inject ions into the QITMS for subsequent
manipulation and detection. Performance of the instrument is demonstrated using a
mixture of ions created by electron impact ionization of perfluorotributylamine (FC-43)
and peptide ions generated by pulsed Cs* bombardment. Resulting data is compared to
those obtained utilizing only the ion trap. Molecular weight, fragmentation, and high
resolution analyses for the sequentially injected mass-filtered peptides show improved
performance over similar measurements employing only the ion trap mass spectrometer.
Performance is optimized when ions are not rf-isolated in the QITMS. Using the hybrid, a
resolution of 33,200 is achieved for angiotensin I. Dramatic reduction of space charge-
induced signal suppression is demonstrated for LSIMS of Glu-fibrinopeptide B. “On-the-
136
fly" collision-induced dissociation is performed for m/z 502 from FC-43, where
fragmentation is induced by increasing the ion injection energy. Collision-induced
dissociation efficiencies for fragmentation of angiotensin I by resonance excitation are
investigated as a function of cooling time for different modes of operation of the hybrid. A
current limitation of the instrument is the time required to port the data for acquisition.
4.2 Introduction
The ability to structurally characterize molecules contained in mixtures has been
greatly simplified by combining two or more mass analyzers. The first mass analyzer
(MS-1) functions as a device to separate ions of interest from the mixture and to pass the
ions into a reaction or activation region. Fragmentation may be induced by depositing
sufficient vibrational or electronic energy into the ion using collisional activation (1),
surface activation (2), or photoactivation (3) methods. The resultant dissociation products
may be analyzed in the second mass analyzer (MS-2) to reveal structurally important
features. Although most common instruments utilized for mixture analysis consist of two
_ mass analyzers separated in space, multiple stages of mass spectrometry are feasible by
separating ion selection, activation, and product analysis in time employing ion trap mass
spectrometers (4). Most commercially available instruments capable of performing
mixture analysis are based on mass analyzers of the same type (5-7), but numerous
examples of hybrid instruments have appeared in the literature (8-13). Hybrid instruments
may afford extension of mixture analysis capabilities by combining the strengths of two
different types of mass analyzers.
137
Due to their versatility and ion storage capabilities, quadrupole ion trap mass
spectrometers (QITMS) have been used in a number of different hybrid configurations.
Magnetic sector instruments have been employed as MS-1 with ion trap mass
spectrometers utilized as MS-2 (14-17). A disadvantage of the sector hybrids is that the
second stage of the mass spectrometer must be floated to high voltages; thus quadrupole
mass filters (Q) were investigated as MS-1 candidates for various tandem combinations,
notably triple quadrupole configurations (18, 19). Quadrupole mass filters have been
interfaced to quadrupole ion traps to select and transmit a single m/z value to the ion trap in
a Q/ion trap/Q configuration (20, 21). A Q/Q/ion trap configuration, where the first
quadrupole was utilized as a selective mass filter and the second quadrupole operated in rf-
only mode as a beam transmitter, was employed to obtain structural information for
filtered molecules. The voltage at which the ion trap electrode assembly was floated
relative to ground was raised to increase the kinetic energy of the ions and to enhance ion
dissociation upon injection into the ion trap (22, 23). Fragmentation may also be induced
in a quadrupole ion trap when resonance excitation (24) is utilized; thus the ion trap may
function as both collision cell and MS-2.
Although multiple stages of mass spectrometry may be performed within the same
collision cell of a quadrupole ion trap, a limitation of utilizing the quadrupole ion trap as
the only mass analyzer is its dynamic range. Typically, no more than ~10° ions may be
trapped before space charge distorts the electric fields, causing reduced sensitivity, mass
accuracy, and resolution (25). Shaped excitation pulses (26) and filtered noise field
techniques (27) have been used for notch injection of molecules of interest. Subsequent
reduction in space charge effects has been demonstrated (28). These techniques, typically
utilizing stored waveforms from a function generator applied to the endcap electrodes of
138
the ion trap, have been used for broadband ejection of low mass matrix ions and for the
selected injection of one or more species in a mixture. Notch injection requires a priori
knowledge of the m/z value for the ion of interest or the use of a “pre-scan” to measure the
m/z values of injected ions in order to calculate the resonance frequency of the notch. An
alternative approach is to employ a quadrupole mass filter to pre-process the ion population
by “step scanning” the quadrupole over narrow mass windows in order to. sequentially
transmit components of a mixture into an ion trap for further manipulation.
This chapter describes the assembly of a quadrupole mass filter/quadrupole ion trap
" mass spectrometer for the analysis of mixtures of molecules. The quadrupole ion trap
filled with helium serves as both collision cell and MS-2. The quadrupole mass filter may
be used either in a mass-selective mode to inject ions of one m/z or in a scanning mode to
' sequentially inject ions into the QITMS. The concept is demonstrated using ions generated
from electron ionization of the model compound perfluorotributylamine (FC-43) and
utilizing a pulsed Cs* source to ionize selected peptides.
4.3 Experimental
The instrument constructed for this work was a hybrid quadrupole mass
filter/quadrupole ion trap mass spectrometer (Q/QITMS), a modification of an ion trap
mass spectrometer that has been previously described (29). The hybrid instrument was
assembled from components of an ITMS quadrupole ion trap mass spectrometer
(Finnigan MAT, San Jose, CA, USA) and a TSQ70 triple quadrupole mass spectrometer
(Finnigan MAT) as illustrated in Figure 4.1.
139
Figure 4.1: Diagram of the Q/QITMS. The EI source is easily replaced by a matrix-
assisted laser desorption ionization source, a pulsed Cs* source, or an electrospray
ionization source. Ions were extracted with a 3 kV lens and were detected with a 20 kV
collision dynode and an off-axis electron multiplier.
140
yNoI
SulIos3t1L,
SoTuO9a[F ro SOUOTII[A
SVL Lad OSL
JorpdnynyAl won|
gjodnipend
— <
[aaww C
apoudq] UOISIOAUOZ) l
' suay de1y uoy sua] QOINOS UOHeZIUCT
4.5) Pasig Jo
youduy uonse[q
uolgoy JozATeuy
141
4.3.1 Ion Source
An electron impact (EI) ionization source (Finnigan MAT) was interfaced to the
Q/QITMS and an electron beam with an energy of 70 eV was used to ionize gas phase
molecules produced by FC-43. A modification of this source incorporated a power supply
and pulsed injection system to produce a 6 keV cesium ion beam for ionization of samples
by liquid secondary ion mass spectrometry (LSIMS) (30). Samples were loaded onto a
probe tip held at ground. Ions formed in the differentially pumped ion source region were
then focused by an einzel lens into the quadrupole mass filter. Typical lens voltages were
-8.4 V, -130.3 V, and -7.5 V, respectively.
4.3.2 Mass Analyzers
The quadrupole mass filter (MS-1), a focusing lens, and the quadrupole ion trap
electrodes (MS-2) were aligned on an optical rail located in the low-pressure region of the
baffled and differentially pumped vacuum manifold as illustrated in Figure 4.1. The
quadrupole rods were hyperbolic high mass rods from a TSQ70 triple quadrupole and had
a nominal mass range of 4000 u. A potential of -5 V was applied as an offset to the
quadrupole. The focusing lens, held at 4 V, was followed by a gating tube lens (+/- 200 V)
and served to focus the exiting ions into the entrance aperture of the ion trap endcap
electrode. The ion trap electrode assembly was floated with a variable dc potential to
control the ion injection energy, typically set to -10 V for FC-43 and -8 V for peptides.
These values correspond to the conditions for maximum ion intensity observed across the
mass range. The ion trapping volume was pressurized with helium to an uncorrected
142
gauge reading of 5.5 x 10* Torr as measured by a Convectron gauge located on the outside
of the vacuum manifold. The rf and dc voltages applied to the quadrupole to establish ion
trajectories were controlled by a DECStation 2100 interfaced to the TSQ70 electronics.
The application of the rf voltage to the ring electrode of the QITMS has been described
previously (29). Sinusoidal auxiliary signals from the ITMS frequency generator were
applied to the endcap electrodes to enable resonance ejection and resonance excitation (31).
A typical QITMS scan function used to obtain molecular weight information is
shown diagramatically in Figure 4.2 (solid lines), along with a quadrupole scan (dashed _
lines). The horizontal axis represents time and the vertical axis represents m/z. The
QITMS scan function consisted of an ionization period, during which the gating tube lens
remained in the “open” position to allow ions into the trap for collection, of 10 - 20 ms.
The ionization period was followed by a variable cooling time. The mass-selective
instability mode of operation for the QITMS (32) was applied to resonantly eject the
trapped ions using a supplementary signal at 520,311 Hz and 4 V (peak-to-peak, endcap-
to-endcap) for ions generated by EI and a signal at 119,936 Hz and 8.8 V (peak-to-peak,
endcap-to-endcap) for ions generated by pulsed Cs*. Ramping time for ejecting the ions
was ~108 ms and was fixed in the ion trap firmware. The amplified signal was displayed’
on the ITMS Compac 386 PC, then transported to the DECStation 2100 running ICIS
software for acquisition and data processing. Resonance excitation was accomplished by
reverse-then-forward rf-isolation of the ion of interest followed by a 30 ms excitation time.
A variable cooling time was applied prior to mass analysis.
143
4.3.3 Synchronization
Two different scanning methods were employed to sequentially inject ions into the
ion trap and are illustrated in Figure 4.2. Figure 4.2(a) shows the "continuous scanning"
method where the quadrupole and ion trap both scan over the same range of m/z values.
The thick line overlaying the quadrupole scan indicates the range of m/z values that were
transmitted by the quadrupole during the ionization period of the ion trap. Only these m/z
values were detected. Depicted in Figure 4.2(b) is the "step scanning" method of
operation. A TTL signal from the quadrupole occurring at the beginning of the quadrupole
scan was used to trigger operation of the 8086 microprocessor on the SAP board of the ion
trap (33). An instrument control language (ICL) procedure was written to scan the
quadrupole over a limited mass range, typically 10 u wide bins, then increment the bin
center by 10 u. Again, the thick line overlaying the quadrupole scan represents the range of
detectable m/z values. Sequential m/z values may be transmitted to the ion trap for
detection, and by a judicious choice of scan bin and step size, every m/z can be transmitted
into the ion trap and detected. The time axis is slashed to represent the time required for
data porting (see below). For both scanning methods, the ion trap electronics were set to
display and port one ion trap "microscan” for each quadrupole bin scan. Mass spectra
were first displayed on the PC then ported to the DECStation 2100, displayed using a
modification of the SPEC data display program, then acquired to the hard disk. The data
porting time was on the order of 450 ms (Figure 4.2) as compared with the actual ion trap
scan time of ~130 ms. The ITMS microprocessor does not continue with its next
instruction until the data from the ITMS have been completely ported to the DECStation
2100; thus the minimum bin scan time, limited by the data porting time, was
144
Figure 4.2: Diagram of scanning modes investigated for injection of ions into the ion trap.
(a) "Continuous scanning" method where the quadrupole mass filter and the ion trap both
scanned over the same mass range. The time delay between the ion trap scan functions
resulted from the data porting and acquisition time. (b) "Step scanning" method where the
quadrupole was scanned over a narrow mass range (bin) while the ion trap was scanned
over the full mass range. Both mass analyzers were synchronized to start scanning at the
same time. The quadrupole scan bin was sequentially stepped to higher m/z values until
the full mass range was covered. The slash in the time axis represents the data porting time
delay.
145
deiyj, uoy ou poyofuy pue gjodnipend £q poytursues L2/u joosuey
uvos sjodnipent) ———
uonouny Ueos SILT
/ L
Tf
2jut
®Q _+ Zul SuUUNY-d014 dey, Uoy puv ajodnapeng) y 146 experimentally determined to be 570 ms. This minimum time required the PC and 1000 to 2000 u to observe Glu-fibrinopeptide B. 4.3.4 Sample Preparation Perfluorotributylamine (FC-43) (Ultra Scientific, N. Kingstown, RI) at a pressure probe tip. 147 4.4 Results and Discussion ‘The objective of this work was to study methods for sequential ion injection into selected peptides. 4.4.1 Ion Injection into the Ion Trap FC-43 typically fragmented in the source region upon electron impact ionization. A scans of the quadrupole mass filter, indicating that all of the ions were injected. Not all 148 Figure 4.3: Selected ion chromatograms resulting from sequential injection of ions bins and triggering QITMS operation after the beginning of the quadrupole scan. 149 a) UNSYNCHRONIZED quadrupole: 50-650 u, 4 sec m/z 69 bak. ve | 100.5 * * J}, | 0 Oi, LAA ‘A 1009 m/z | 1 L. of | | | 504 100 150 200 100, m/z 69 1007 m/z 264 . A 50; a ai nm 1007 m/z 414 100, m/z 502 | 507 Scan Number 150 single scans of the quadrupole mass filter, however, transmitted all of the FC-43 ions. The result of using the step scanning method is shown in Figure 4.3(b). The The selected ion chromatograms resulting from the synchronization of the mass As demonstrated by Julian and Cooks, the use of broadband excitation with frequency notches to eject matrix ions from the trap can significantly improve the signal-to- 151 noise (S/N) ratio for the analyte (28). An analogous experiment illustrated in Figure 4.4 Figure 4.5 illustrates the typical performance of the instrument for a mixture of ions were markedly improved and the chemical background usually detected when a liquid 152 Figure 4.4: Reduction in signal suppression achieved utilizing mass-selected sequential chromatogram is shown as an inset. 153 RF-ONLY INJECTION a) oe b) 500 1000 1500 500 1000 1500 wer |e 154 Figure 4.5: Analysis of a mixture of peptides from the human angiotensin family with a bin scan width of 2 u resulting in a 22.5 second scan time over the mass range. 155 0? Jequiny ues 002 Ost oot OrT O6e l | ybog aseg r OC vez | | OZ LOOT 069°¢ | CeO -2/u L OZ (9 ZU ] ulsusj}orsue II Ulsusjorsue-[, Je A] If Ulsuojorsue-[ eA] | OV 09 108 9b0T I] Ulsua}OIsUe NOLLOALNI TVILNANOAS 0osT OOOT | 00S L6cl jursuajorsue | | Zt msuajoysue-yea} BO! _ LOS 001 0c 08 9P0l_ NOLLOGENI AINO-AY OOT (v soULpUNGY danepey 156 matrix is utilized is not seen. Post-source fragmentation was observed resulting from Since complex mixtures of ions may contain components with similar mass-to- the transmission overlap. 4.4.2 Mass Resolution Space-charge effects typically provide an upper limit for resolution on the QITMS number of extraneous ions injected into an ITMS. In this work we use the quadrupole 157 _mass filter in selected-ion injection mode to minimize the number of unwanted ions The high-resolution performance of the Q/QITMS for the analysis ‘of large The addition of a cooling time prior to mass analysis affords the damping of kinetic operation of the QITMS and hybrid. In the first set of experiments, the quadrupole was 158 Figure 4.6: High resolution mass spectrum of human angiotensin I (MW 1296 u) ions from the trapping volume. Relative Abundance 159 100+ l2 5 ne Oe Se A 1296 1297 1298 1299 1300 1301 160 operated in rf-only mode and reverse-then-forward rf-isolation scans were performed in injection mode and ions were then rf-isolated in the ion trap. An average mass resolution of 12,820 was calculated. Similar results (m/Am=13,640) were achieved when ions were selectively injected but not rf-isolated in the ion trap. Figure 4.7 displays a "least squares Simulations of ion motion inside the trapping volume indicate that initial conditions trajectories of the population of the m/z of interest when adjacent ions are resonantly ejected 161 Figure 4.7: Linear regression analysis of the calculated resolution for m/z 502 from FC- mass-selected injection with rf-isolation in the ion trap, A indicates QITMS only. 162 — (D9SU) SUL], SUI[OOTD) | | | | { | | | | 4 i} l Lj | i} ' i i} $ i —+ QOOTT + OOSTT 00szl OOOCT r OOSET *~ QOOFT UOTN[OSdy 163 (42). Yost et al. observed that non-linear effects are intensified as ion storage time is of operation shown in Figure 4.7 corroborates this observation. 4.4.3 Tandem Mass Spectrometry The next set of studies examined the potential to perform MS/MS analysis "on the voltage at which the molecules of interest may be optimally trapped as well as the value at 164 Figure 4.8: “On-the-fly” fragmentation of ions generated from FC-43. Decrementing the peaks result from the sequential injection of m/z 264. Relative Abundance 807 165 100] m/z:264 60 | 20 | 0 an _ E+06 100 |} m/z:502 60 7 20 4 _ E+06 Float Voltage p fotlalbadlallalabtllldod essed all ddl re re 24 30 166 which dissociation is most facile. This method did not produce significant dissociation for An auxiliary sinusoidal signal is typically placed across the endcap electrodes to The amino acid sequence of the peptide is given in Figure 4.9 with the expected m/z and y-type ions that the sequence of the peptide could be deduced from the data. The signal 167 Figure 4.9: MS/MS of human angiotensin I by resonance excitation. The rf level on the Ions were not subsequently rf-isolated in the ion trap. 168 116 272 371 534 647 784 *881 1028 1166 1279 b-type 1200 - 1400 1400 | 169 at m/z 1309 indicated by a filled circle is thought to result from the creation of adducts due The spectrum in Figure 4.9(b), by comparison, results from mass-selected injection spectrum rich in sequence information. 170 The efficiency of fragmentation induced by resonance excitation of angiotensin I of nonlinear resonance conditions on the expanded ion cloud (38). 171 4.5 Conclusion A quadrupole mass filter/quadrupole ion trap mass spectrometer, an instrument limited by the time required to port the data from the PC to the DECStation 2100. 172 1. Jennings, K. R. (1968) Int. J. Mass Spectrom. Ion Phys. 1227-235. 2. Mabud, M. A., Dekrey, M. J., and Cooks, R. G. (1985) Int. J. Mass Spectrom. Ion 3. Griffiths, I. W., Mukhtar, E. S., March, R. E., Harris, F. M., and Beynon, J. H. 4, Louris, J. N., Brodbelt, J. S., Cooks, R. G., Glish, G. L., Van Berkel, G. J., and 5. Yost, R. A., Enke, C. G., McGilvery, D. C., Smith, D., and Morrison, J.D. (1979) 6. Schey, K., Cooks, R. G., Grix, R., and Wollnick, H. (1987) Int. J. Mass Spectrom. 7. Tomer, K. B., Guenat, C. R., and Detering, J. (1988) Anal. Chem. 60 2232-2236. 9. Gaskell, S. J., Reilly, M. H., and Porter, C. J. (1988) Rapid Commun. Mass 10. Taylor, L. C. E., and Poulter, L. (1989) in Advances in Mass Spectrometry 11. Schoen, A. E., Amy, J. W., Ciupek, J. D., Cooks, R. G., Dobberstein, P., and Jung, G. (1985) Int. J. Mass Spectrom. Ion Proc. 65 125-140. 173 12. Beaugrand, C., Devant, G., Nermag, S. N., and Janoven, D. (1986) Proc. of the 13. Jennings, K. R. (1983) Proc. of the 31st ASMS Conf. on Mass Spectrom. and 14. March, R. E., and Hughers, R. J. (1989) Quadrupole Storage Mass Spectrometry, 15. Ho, M., Hughes, R. J., Kazdan, E., Matthews, P. J., Young, A. B., and March, R. 16. Suter, M. J.-F., Gfeller, H., and Schlunnegger, U. P. (1989) Rapid Commun. Mass 17. Schwartz, J. C., Kaiser, R. E., Cooks, R. G., and Savickas, P. J. (1990) Int. J. 18. Fraefel, A., and Seibl, J. (1985) Mass Spectrom. Rev. 4151-221. 19. Yost, R. A., and Enke, C. G. (1983) in Tandem Mass Spectrometry (McLafferty, 20. Kofel, P., Reinhard, H., and Schlunnegger, U. P. (1990) Proc. of the 38th Conf. on 21. Kofel, P., Reinhard, H., and Schlunegger, U. P. (1991) Org. Mass Spectrom. 26 463-467. 174 22. Cooks, R. G., and Morand, K. L. (1990) Proc. of the 38th ASMS Conf. on Mass 23. Morand, K. L., Horning, S. R., and Cooks, R. G. (1991) Int. J. Mass Spectrom. 24. Louris, J. N., Cooks, R. G., Syka, J. E. P., Kelley, P. E., Stafford, G. C., Jr., and 25. Johnson, J. V., Yost, R. A., Kelley, P. E., and Bradford, D. C. (1990) Anal. Chem. 26. Chen, L., Wang, T.-C. L., Ricca, T. L., and Marshall, A. G. (1987) Anal. Chem. 27. Goeringer, D. E., Asano, K. G., McLuckey, S. A., Hoekman, D., and Stiller, S. 28. Julian, R. K., and Cooks, R. G. (1993) Anal. Chem. 65 1827-1833. 29. Jonscher, K., Currie, G., McCormack, A. L., and Yates, J. R., I (1993) Rapid 30. a) Kaiser, R. E., Jr., Louris, J. N., Amy, J. W., and Cooks, R. G. (1989) Rapid 31. Kaiser, R. E., Jr., Cooks, R. G., Stafford, G. C., Jr., Syka, J. E. P., and Hemberger, P. H. (1991) Int. J. Mass Spectrom. Ion Proc. 106 79-115. 175 32. Stafford, G. C., Jr., Kelley, P. E., Syka, J. E. P., Reynolds, W. E., and Todd, J. F. J. (1984) Int. J. Mass Spectrom. Ion Proc. 60 85-98. 33. Steenvoorden, R. (1994) Personal Communication. 34, Schwartz, J. C., Cooks, R. G., Weber-Grabau, M., and Kelley, P. E. (1988) Proc. 35, a) Bier, M. E., Schwartz, J. C., Schey, K. L., and Cooks, R. G. (1990) Int. J. Mass 37. Schwartz, J. C., Syka, J. E. P., and Jardine, I. (1991) J. Am. Soc. Mass Spectrom. 38. Wu, H.-F., and Brodbelt, J. S. (1992) Int. J. Mass Spectrom. Ion Proc. 115 67-81. 40. Julian, R. K., Reiser, H.-P., and Cooks, R. G. (1993) Int. J. Mass Spectrom. Ion 41. Eades, D. M., Johnson, J. V., and Yost, R. A. (1993)... Am. Soc. Mass Spectrom. 42. Cox, K. A., Williams, J. D., Cooks, R. G., and Kaiser, R. E., Jr. (1992) Biol. 43. Roepstorff, P., and Fohlman, J. (1984) Biomed. Mass Spectrom. 11 601. 176 44, Thorne, J. C., Ballard, K. D., and Gaskell, S. J. (1990) J. Am. Soc. Mass Spectrom. Spectrom. 8 953-956. 177 Chapter 5 High Sensitivity Peptide Mixture Separation Using Low- - 5.1 Overview The use of low-flowrate electrospray ionization (microspray) for the analysis of detected by CE-MS on the LCQ ion trap. Crude peptide separations were performed using 178 step elutions from a pre-concentration membrane. Limits of detection for angiotensin were technique for the analysis of complex mixtures of peptides. 5.2 Introduction Liquid chromatography coupled to mass spectrometry (LC-MS) is increasingly technique is that lowering the flowrate causes an increase in elution peak widths thus 179 increasing the concentration limit of detection and decreasing the detectability of low - abundance species. CE-MS (12-16) has been proposed as an alternative to LC-MS for the high This technique increased the utility of CE-MS for the analysis of biological samples. An electrical contact is required for the establishment of the CE field and the Mingling the sample with a charged organic coaxial sheath liquid at the needle tip has been 180 widely used to transfer charge to analytes (14). Careful selection of sheath composition transfer line has been reported to eliminate interaction effects (11). The work discussed here details the development of a novel microspray ionization from a hydrophobic membrane with varying ratios of buffer/solvent is proposed as a 181 means of concentrating and simplifying peptide mixtures prior to CE-MS analysis and preliminary data is presented. 5.3 Experimental 5.3.1 Ion Source A microspray ionization source was constructed utilizing a lucite sample stage for microspray ionization. Conditions for CE experiments are described below. Figure 5.1 also includes a diagram of an electrospray source that was modified and capillary reduces charging problems that may be encountered with glass capillaries (22). 182 Figure 5.1: Diagram of microspray ionization source. The heated capillary and lensing the hybrid instrument Q/QITMS. 183 OUI] JaJsuely, (— mats dT UINUTCId JOWIWTYS | 9A99[S SULIB[NSUT asUyOA la If of \ a8v}S a[duies loyejndimuey 7 AX 184 The end of the capillary was drilled out to a conical shape to allow the microspray needle to Lens 2 1.4 V, and Lens 3 -9.4 V. 5.3.2 .Needles and Liguid Junctions Several types of microspray needles and liquid junctions were investigated and are schematically illustrated in Figure 5.2. 185 5.3.2.1 Micropipette Needles with Liquid Junction Needles were made by heating 1 um OD x 0.5 wm ID borosilicate glass prior to the application of epoxy, illustrated in Figure 5.2(a). 5.3.2.2 Pulled Capillary Needles Needles were constructed as described by Davis et al. (11). A ~30 g weight (large . pulled into a needle. A scribe was used to cut the tips of the pulled capillary needles under were washed with methanol before use to test the patency. 186 Figure 5.2: Needle and liquid junction configurations used in this work. (a) A pulled silica needle pushed into the ends. b) Pulled Capillary Needle 187 Tip of Pulled Glass - pane Platinum wire Fused Silica Na Cc) —. Metal Union : Fused Silica ee ™~ Platinum wire 188 5.3.2.3 Metal Union Voltage may be applied to a liquid sample by connecting two pieces of fused silica union using HPLC fittings. 5.3.2.4 Teflon Junction A novel liquid junction was developed employing a 1 cm length of 125 um mm, providing dead volumes of 30 - 100 nL. 189 5.3.3 Mass Spectrometry Two mass spectrometers were employed in the work described here. The hybrid 1.6 kV. A newly commercialized ion trap mass spectrometer, the LCQ (Finnigan MAT), stable number of ions in the trap and eliminate space-charge effects. The AGC target 190 Figure 5.3: Diagram of the LCQ ion trap mass spectrometer. 191 Jad yA] W0.1999 107 ayes) UOT ajodo0}IQ ATUO-yy dei, uoy ajodnapend k repides paeoyy 192 values were 6 x 10’ ions for full scans, 1 x 10° ions for MS" experiments, and 1 x 10’ ions for high resolution scans. 5.3.4 Chromatography 5.3.4.1 Capillary Electrophoresis CE columns were prepared using the protocols of Bruin and co-workers (23) and to be 190 nL/min. Columns were flushed with water and buffer for ~1 hr prior to use. 5.3.4.2 Membrane Chromatography A ~0.5 cm length of 300 um ID Teflon tubing (Chromtech, Apple Valley, MN, of stainless steel wire to create a membrane cartridge as illustrated in Figure 5.4. Teflon 193 Table 5.1: Protocol for preparing CE columns. Reagent Reaction Helium acetone 0.3 100 194 membranes impregnated with poly(styrenedivinylbenzene) coated beads (Empore (365 um OD x 50 um ID) was placed at either end of the cartridge. Membranes were activated by washing the membrane in the cartridge with 10 nL buffer. 5.3.5) Sample Preparation Human angiotensin I (MW 1296 Da, Cat. No. A-9650, Lot. No. 13H59101), C-6780, Lot No. 93H9554) were purchased from Sigma Chemical Co. (St. Louis, MO, 195 Figure 5.4; Diagram of Teflon membrane cartridge used for separation of peptide mixtures. 196 Kavyydes voyis pasng JUBIGUII]A, suIqn |, WOTJOL, 197 USA) and used without further purification. TPCK-treated porcine trypsin (Cat. No. microspray infusion experiments were further diluted with methanol in a 1:1 mixture. 5.4 Results and Discussion The objectives of this work were to develop a robust microspray ionization source was used to probe various separation techniques. 198 5.4.1 Microspray Needle Development Two types of microspray ionization needles are typically in use. The first utilizes Needle lifetimes were typically on the order of 3-4 days. - An illustration of the sensitivity of the technique for microspray infusion is shown nL/min. Stable spray was not attainable for less concentrated solutions or lower flowrates. 199 Figure 5.5: High sensitivity microspray infusion of a mixture of angiotensin I and microscans, representing the consumption of 75 amol of material. Relative Abundance 200 melittin 400 600 800 1000 1200 1400 201 The mass spectrum depicted in Figure 5.5 represents the consumption of 75 amol of range for this experiment (data not shown). A qualitative comparison of performance between the glass micropipette needles were interfaced to the Teflon membrane cartridges, discussed below. 5.4.2 Liquid Junction Development As discussed above, there are several methods whereby voltage can be applied to a time-consuming leak testing. Metal unions using epoxy must be disposed of when the 202 needles block, which can be as often as every 1-8 hours (8, 11). In addition, high voltage electrode to induce an arc. This significantly compromises the spray stability. A unique liquid junction has been designed which incorporates a 125 um diameter unions (data not shown). 203 5.4.3 Separation of Peptide Mixtures The analysis of biochemical systems typically involves the analysis of mixtures of peptide mixtures. 5.4.3.1 Neutral Loss Scan Neutral loss scanning is a technique commonly used on triple quadrupole mass of phosphopeptides in a mixture. Microspray ionization using a pulled glass micropipette tip with a platinum wire were investigated, the peptide with m/z 693 and that with m/z 831. The quadrupole mass 204 filter was set to transmit ions within a 2 u mass window around 693 and 831, respectively. the peak at m/z 781 results from the neutral loss of 49 u from the precursor at m/z 831. 5.4.3.2 Separation by Capillary Electrophoresis CE is a powerful and sensitive technique for analyzing simple mixtures of ionization sources. Preliminary data is presented here from our first attempts at interfacing CE to a instrument makes it easy to perform relatively complex experiments. For example, Figure 205 Figure 5.6: Mass spectra arising from neutral loss scanning of the Q/QITMS. Ions were 781.5. 206 0¢8 008 z/ul OL z/u row ST8Z 0¢ OV 09 . | 08 | OOT + OC OV + O9 + 08 OOT (8 207 5.7 demonstrates an MS* experiment on an ion with m/z 880 from a tryptic digest of o- use provided by the upgraded software. Aminopropyltrimethoxysilane was covalently linked to silane groups on the walls and 579 result from impurities due to solvent filtering. Subsequent experiments have been 208 Figure 5.7: MS‘ on the doubly-charged ion of m/z 880 from a tryptic digest of the model low mass end of the fragmentation spectrum. Results are displayed in the bottom panel. 209 o| #8 ,SIN 715] 9/1 BE Ge fe es A (SIN SW (v qd 210 1383.1 b) 1266.4 979.6 879.8 MS 747.7 800 100- 80- 60- 1200 1400 1600 1000 600 400 +1 i k--— 13 "4 soUBpUNgY dajeay 450 400 350 300 250 200 150 bt! +] @436 @266 100- | 880 Ms* 80- 250 200 150 100 211 Figure 5.8: Mass spectrum and selected ion chromatogram resulting from injection of 1 To provide the mass spectrum, 23 scans were summed. Relative Abundance 100 5 80 5 60 5 40 - 20 - (M+3H)*3 443.4 212 50 { (M424) jun Mul 100 300 500 Scan Number 600 700 800 900 1000 213 performed without solvent filtering, eliminating this effect. The width of the elution peak is preliminary results. 5.4.3.3 Separation By Membrane Chromatography . One limitation of CE is that relatively concentrated samples are required. analysis by CE. 5.4.3.3.1 Sensitivity Peptides typically bind to hydrophobic surfaces. ‘They can be released from these for binding and the less hydrophobic peptides are released. Differential release is obtained 214 by changing the relative amount of organic in the solvent. Most research using pre- might provide more sensitive performance. The calculated limit of detection based upon the logarithmic fit to the data was 1.3 fmol/uL solution. These preliminary results suggest that the hydrophobic membranes 215 Figure 5.9: Mass spectrum of 10 fmol of angiotensin I obtained from a 10 uL of a 1 and 20 scans were summed to provide the mass spectrum. Relative Abundance 1007 80 | 20 7 60° AQ | 216 (M+3H)*? (M+2H)*? 10 fmol angiotensin I loaded onto membrane 627.8 718.3 782.6 400 600 — 800 1000 1200 m/z 217 - Figure 5.10: Calculated signal-to-noise ratio vs. amount of sample loaded onto a possibility of sample losses at low sample concentrations. 218 OOOT (jowy) pepo] ajdures Ol Ol 0¢ Oc . oney N/S payemnoayeD 219 provide a sensitive concentrating device that interfaces well with the Teflon junction system detection. Air bubbles have often been observed forming on the syringe side of the eliminated before testing the performance of the system using the micropipette needles. 5.4.3.3.2 Mixture Simplification Peptides obtained from a tryptic digest of o-casein were separated by CE (data not _ prior to CE analysis was investigated. A 0.5 uL aliquant of a 1 pmol/L solution of the the peptides identified. The sample of o-casein obtained from Sigma is contaminated with 220 Figure 5.11: Amino acid sequences and observed peptides for o—casein. Tryptic peptides performed to determine charge states and aid in identification of observed ions. 51 101 151 201 221 Casein Type 1, MW 24529 u, 214 a.a. MKLLILTCLV AVALARPKHP IKHQGLPQEV LNENLLRFFV APRPEVFGKE I 1 I Ms 1 Ti, Ti, Ti6p Tyg i { i i I i C C IGVNQELAYF YPELFROFYQ LDAYPSGAWY YVPLGTOYTD APSFSDIPNP 21 ~ Casein Type 2, MW 26019 u, 222 a.a. 51 101 151 201 MKFFIFTCLL AVALAKNTME HVSSSEESII SQETYKQEKN MAINPSKENL NOFYQKFPQY LQYLYQGPIV LNPWDQVKRN AVPITPTLNR EQLSTSEENS KKTVDMESTE VFTKKTKLTE EEKNRLNFLK KISQRYOKFA LPQYLKTVYQ HOKAMKPWIO PKTKVIPYVR YL 222 another type of casein, designated here as type 2. Approximately 58% of the amino phosphopeptide was observed using the two different ion traps. Crude separation of peptides at different concentrations of methanol was could be performed during the course of an elution. 5.5 Conclusion A microspray ionization source has been developed for the high sensitivity liquid junction was designed for ease of 223 Table 5.2: Dominant ions observed at 30%, 50%, and 70% methanol during step Percent Methanol Observed m/z 70 693, 980 224 Table 5.3: Percent relative abundance of selected ions when eluting a casein digest peptide mixture isocratically from a hydrophobic membrane with 1:1:0.5 methanol:water:acetic RT m/z m/z m/z m/z m/z m/z m/z m/z 2 66.84 13.72 40.14 34.66 100 0 41.24 0 3 84.81 24.88 7146 28.1 100 22.06 64.44 0 4 100 53.66 88.72 26.8 46.12 35.26 97.2 26.35 * - RT Int. refers to a 30 second retention time interval beginning at a retention time of 2.5 min. All 225 use and to eliminate background derived from interactions of sample with metal and junction has not yet been investigated. Neutral loss scanning on a triple quadrupole mass spectrometer is a common spectrometer to identify phosphopeptides in a mixture. Hydrophobic membranes have shown great promise as pre-concentration devices membranes will be investigated to add other chromatographic dimensions. 226 - 5.6 References 1. Gelpi, E. (1995) J. Chromatogr. A 703 59-80. 2. Meng, C. K,, Mann, M., and Fenn, J. B. (1988) Z. Phys. D. - Atoms, Mol. Clusters 3. Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F., and Whitehouse, C. M. (1989) 4. Fenn, J. B., Mann, M., Meng, C. K., and Wong, S. F. (1990) Mass Spec. Rev. 9 5. Fenn, J. B. (1993) J. Am. Soc. Mass Spectrom. 4524-535. 6. Emmett, M. R., and Caprioli, R. M. (1994) J. Am. Soc. Mass Spectrom. 5 605- 7. Andren, P. E., Emmett, M. R., and Caprioli, R. M. (1994) J. Am. Soc. Mass 8. Valaskovic, G. A., Kelleher, N. L., Little, D. P., Aaserud, D. J., and McLafferty, F. 9. Figeys, D., van Oosteveen, I., Ducret, A., and Aebersold, R. (1996) Anal. Chem. 10. Gale, D. C., and Smith, R. D. (1993) Rapid Commun. Mass Spectrom. 7: 1017- 11. Davis, M. T., Stahl, D. C., Hefta, S. A., and Lee, T. D. (1995) Anal. Chem. 67 4549-4556. 227 12. Olivares, J. A., Nguyen, N. T., Yonker, C. R., and Smith, R. D. (1987) Anal. 13. Lee, E. D., Mueck, W., Henion, J. D., and Covey, T. R. (1988) J. Chromatogr. 14. Smith, R. D., Olivares, J. A., Nguyen, N. T., and Udseth, H. R. (1989) Anal. 15. Johannson, I. M., Huang, F. C., Henion, J . D., and Zweigenbaum, J. (1991) J. 16. Smith, R. D., Wahl, J. A., and Goodlett, D. R. (1993) Anal. Chem. 65 574A- 17. | Tomlinson, A. J., and Naylor, S. (1995) J. Cap. Elec. 5 225-233. 18. Tomlinson, A. J., and Naylor, S. (1995) J. High Resol. Chromatogr. 18 384-386. 20. Severs, J. C., Harms, A. C., and Smith, R. D. (1996) Rapid Commun. Mass 21. Foret, F., Thompson, T. J., Vouros, P., Karger, B. L., Gebauer, P., and Bocek, P. 22. Chowdhury, S. K., Katta, V., and Chait, B. T. (1990) Rapid Commun. Mass 23. Bruin, G. J. M., Huisden, R., Kraak, J. C., and Poppe, H. (1989) J. Chromatogr. 24. Thorsteinsdottir, M., Isaksson, R., and Westerlund, D. (1995) Electrophoresis 16 557-563. 228 25. Wilm, M. S., and Mann, M. (1994) Int. J. Mass Spectrom. Ion Proc. 136 167- 27. Johnson, J. V., Pedder, R. E., and Yost, R. A. (1991) Int. J. Mass Spectrom. Ion Proc. 106 197-212. 229 Chapter 6 Summary Presented in this dissertation are a number of approaches investigated for the chapters. Below is presented future directions for these projects. 6.1 Matrix-Assisted Laser Desorption Ion Trap Mass Spectrometry A number of innovations have been implemented in MALDI/ion traps since the publication in 1993 of the work described in Chapter Three (1). Sensitivity in the low 230 femtomole range has been reported for small peptides using an external injection commercial instrument in the future (9). 6.2 Hybrid Quadrupole Mass Filter/Quadrupole I Ion n trap Mass The hybrid instrument was shown to provide improved performance both in the Finnigan MAT may be employed to investigate the approach. 231 Preliminary data was presented in Chapter Five from a microspray ionization biological processes. 232 1. Jonscher, K. R., Currie, G., McCormack, A. L., and Yates, J. R., II (1993) Rapid Commun. Mass Spectrom. 7 20-26. 4, Mclver, R. T., Jr., Li, Y., and Hunter, R. T. (1994) Proc. Natl. Acad. Sci. USA 91 4801-4805. 6. Doroshenko, V. M., and Cotter, R. J. (1993) Rapid Commun. Mass Spectrom. 7 822-827. 8. Doroshenko, V. M., and Cotter, R. J. (1996) Rapid Commun. Mass Spectrom. 10 65-73. 9. Qin, J., Steenvoorden, R. J. J. M., and Chait, B. T. (1996) Anal. Chem. 68, 10, 1784-1791. 233 Karen R. Jonscher was born on March 19, 1959, in Denver, Colorado, to Max and Colorado, and is expecting the birth of her third child in November of 1996.
[nal
=)
ay
=|
Bad
re
=}
=|
oe;
“en
ome
eS
2.
YQ
+=
DECStation display parameters to be set to very narrow windows with no signal displayed
so that the spectral display time would be minimized. In addition, the windows on the
DECStation representing the status of the instrument (Ze., lens voltages, pressures, etc.)
had to be changed to non-updating windows in order to achieve the minimum bin scan
time above. The scan times for the experiments ranged from 22.5 (scanning from 1000 u
to 1075 u for the case of a mixture of angiotensin II peptides) to 60 s for scanning from °
of 1 x 10* Torr (uncorrected) entered the ion source region through a needle valve and was
ionized by electron impact ionization using electrons with 70 eV of energy. The peptides
[Val*]angiotensin III (MW 917, Cat. No. A6277, Lot. No. 053F58452), angiotensin II
(MW 1046, Cat. No. A9525, Lot No. 13H59001 ), [Val*Jangiotensin Tt (MW 1032, Cat.
No. A2900, Lot No. 119F58101) and angiotensin I (MW 1296, Cat. No. A9650, Lot No.
13H59101) were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and used
without further purification. Peptides were separately dissolved in 0.1% trifluoroacetic acid
to a concentration of 25 pmol/L then mixed together in equal volumes. Approximately
1 uL of 1:1 glycerol:thioglycerol and 0.5 WL of the peptide mixture were co-deposited onto
the gold electroplated probe tip. Glu-fibrinopeptide B (MW 1571, Cat. No. F3261, Sigma
Chemical Co.) was similarly diluted to a concentration of 5 pmol/uL and applied to the
the ion trap and the potential for performing “on the fly” tandem mass spectrometry using
a hybrid instrument configuration of a quadrupole mass filter and quadrupole ion trap mass
spectrometer. Results for the performance of the instrument are reported for FC-43 and
simple mixture of singly-charged ions was produced with monoisotopic mass-to-charge
ratios of 69 u, 131 u, 264 u, 414 u, 502 u, and 614 u (34). Two different quadrupole
scanning methods, continuous and step scanning, were evaluated for sequential injection of
this set of ions as diagrammed in Figure 4.2. For the continuous scanning experiment, the
quadrupole was scanned continuously from 50 to 650 u with a scan time of 4 s and the ion
trap was scanned over the same mass range with a scan time of ~130 ms. Single ion trap
microscans were ported to the DECStation 2100 and acquired. The scans of the
quadrupole and ion trap were not synchronized. Figure 4.3(a) shows the selected ion
chromatograms (SIC) for the above ions with ion intensity plotted against ion trap scan
number. Approximately 30 pscans correspond to one quadrupole full-range scan; thus the
experiment in Figure 43(a) reflects approximately 5-6 scans of the quadrupole mass filter.
Signal was observed for all of the selected ion chromatograms over the course .of several
generated from perfluorotributylamine. The relative injection energy was 10 ev. The
amplitude of the rf voltage on the ion trap ring electrode was Set so as to exclude ions with
m/z less than 40 u from the trapping volume. (a) The quadrupole scanned from 50 u -
650 u in 4 seconds while the QITMS scanned over the same mass range in ~150 ms. (b)
The mass analyzers were synchronized by “step scanning” the quadrupole in 10 u wide
100- ITMS: 50-650 u, 130 msec
A A
100 m/z aI
1005 m/z 502
1005 m/z 614 |
b) | [SYNCHRONIZED] quadrupole: 50-650 u, 10 u bin, 0.6 sec
ITMS: 50 - 650 u, 130 msec
hg tae et
al AA \ h i\
100; m/z 614
This is particularly evident in the chromatogram for m/z 414.
quadrupole mass filter was set to scan from 50 u to 650 u in 10 u wide mass bins, each bin
having a scan time of 600 ms, affording a 36 s scan time over the desired mass range. The
lower limit for the bin scan time was the time necessary to port the data from the ITMS to
the DECStation (450 ms) and was not a function of the QITMS scanning time (130 ms).
If the data porting time were negligible, the scan time over this mass range would be on the
order of 9 seconds. Mass spectra from 200 scans of the ion trap were acquired.
analyzers, shown in Figure 4.3(b), are strikingly different from those obtained in Figure
4,3(a) where the two mass analyzers were free-running. Using the "step scanning"
technique, mass separation of the FC-43 fragmentation products by the quadrupole clearly
afforded sequential injection of ions, from low to high m/z values, into the ion trap. The
peaks of smaller intensity in the chromatogram for m/z 264 result from fragmentation of
the mass selected m/z 502 ion. The ion chromatogram for m/z 131 shows a double-peak
pattern; the peak at the lower scan number results from mass selection of m/z 131 and
produces a corresponding fragment ion of m/z 69, while the peak at the higher scan
number results from dissociation of mass-selected m/z 197, producing fragment ions of
m/z 69 and m/z 131. The ion chromatogram for m/z 69 shows a quadruple peak pattern
deriving from mass selection of m/z 69 as well as fragmentation from m/z 131, from m/z
181, and from m/z 197.
compared S/N ratios for Glu-fibrinopeptide B ions generated by pulsed Cs* bombardment
and injected into the ion trap using no pre-processing with those obtained using step
scanning. As shown in Figure 4.4(a), when an rf-only potential was applied to the
quadrupole, the ion trap was quickly filled to the space-charge limit due to the transmission
of the ion background generated by desorption of the matrix. The peptide signal was
suppressed. Figure 4.4(b) depicts the mass spectrum, with the SIC displayed as an inset,
obtained by the step scanning injection of the same peptide. The mass filter was scanned
from 1000 to 2000 u in 10 u wide mass bins with a bin scan time of 0.6 s, a 60 s scan over
the entire mass range. Matrix suppression effects were dramatically reduced and a S/N
ratio of 17.2 was calculated for m/z 1572.
peptides with similar structures. An equimolar mixture of angiotensin I, angiotensin II,
[Val*]angiotensin III and [Val*]angiotensin II was applied to the probe tip and ionized by
Cs* bombardment. Depicted in Figure 4.5(a) is the mass spectrum resulting from rf-only
transmission of ions through the quadrupole and injection into the ion trap. The appearance
of the mass spectrum is similar to that obtained using only the QITMS as a mass analyzer.
The mass spectrum is complicated by signals resulting from fragmentation of the
precursor ions as well as matrix clusters and adducts. By comparison, Figure 4.5(b)
depicts the result of sequential injection of the peptide mixture using the quadrupole in step
scanning mode. The quadrupole mass filter was stepped from 850 to 1350 u using 5 u
wide bins and a bin scan time of 0.6 s. A total of 110 scans were summed, corresponding
to 1 full mass range cycle of the quadrupole. The S/N ratio and resolution of the precursor
injection. The QITMS rf exclusion limit was set to exclude ions with m/z below 100 u
from the trapping volume. The gating time to allow ions into the trap was 80 ms. (a)
Hybrid operated in rf-transmission mode with all ions above 10 u transmitted through the
quadrupole and injected into the ion trap for mass analysis. Complete signal suppression
was observed for Glu-fibrinopeptide B. (b) Sequential injection by “step scanning” the
quadrupole from 1000 u - 2000 u significantly improves the S/N ratio. The selected ion
100 7
60 7
1450 1550 1650
100 1 | SEQUENTIAL INJECTION (M+H)t
m/z 1571
807
60 7
“20” 60.” 100
Scan Number
20
generated by LSIMS. The QITMS gating time was 20 ms and the rf exclusion limit was
100 u. (a) Hybrid operated in rf-transmission mode. (b) “Step scanning” the quadrupole
from 850 u - 1350 u serves as a mass chromatography stage. 110 scans representing one
full mass range cycle of the quadrupole were summed. (c) Chromatographic separation of
m/z 1032 and m/z 1046. The quadrupole was stepped from 1000 u to 1075 u in 2 u steps
SO+H
t OV
t O9
r 08
LOOT
cota
| Op
SO+H
OF
| 09
L 08
LOOT
00ST 0001 00s
NN
[If Ulsusjorsue-[ eA]
(q
Ov
09
I[ Ulsudjorsue
collision-induced dissociation with the helium bath gas or surface-induced dissociation
(35).
charge ratios, it was of interest to investigate the separation resolution of the sequential
injection technique. Illustrated in Figure 4.5(c) is an example of the mass chromatographic
separation of [Val*]angiotensin IT (MW 1032) and angiotensin II (MW 1046). The
selected ion chromatograms clearly demonstrate the ability of this technique to mass
separate species within a relatively narrow range of m/z values. There is a small region of
overlap where both ions are being simultaneously transmitted as evidenced in the base peak
chromatogram. Increasing the resolution of the quadrupole to narrow the mass
transmission window may serve to reduce the extent of simultaneous transmission.
Reducing the width of the quadrupole scan bin and decreasing the step size did not reduce
(36) and the primary cause of space-charge is the number of ions in the trap. Selected
injection of ions serves to reduce space-charge and improve resolution, especially for
components in a mixture. Notch injection using shaped excitation pulses (28), as well as
the application of filtered noise fields (27), has been successfully employed to minimize the
injected into the ion trap.
molecules was investigated with the model peptide angiotensin I (monoisotopic MW
1295.6). The rf exclusion limit of the ion trap was set to 100 u and the quadrupole
transmitted ions by scanning a 10 u window around m/z 1296. The de offset controlling
the resolution of the quadrupole was set to 20% of the maximum offset. The resolution on
the quadrupole cannot be calculated since the peak width is dependent upon the ion trap
resolution. Typically, 30 microscans were averaged then ported as one scan to the
DECStation. Illustrated in Figure 4.6 is a high resolution mass spectrum for m/z 1296
from angiotensin. The spectrum was obtained by slowing the mass scan speed by a factor
of 200 employing the technique developed by Schwartz et al. (37). The mass spectrum
represents data from a single QITMS microscan in which a resolution of 33,220 was
calculated. The average resolution obtained for 30 microscans was typically half of this
value, resulting from peak shifting that occurs in this type of experiment. The average
resolution obtained for 30 microscans utilizing the QITMS was similar to that obtained
using the hybrid instrument and mass-selecting ions with the quadrupole. Mass resolution
in this range is more than sufficient for most biochemical applications.
energies and trajectories of trapped ions. Compacting the ion cloud near the center of the
trapping volume increases the detection efficiency and reduces the amount of time required
to eject the ions, leading to improved resolution (38). In a series of experiments, the effect
of cooling time on resolution of m/z 502 from FC-43 was studied for different modes of
obtained by slowing the QITMS acquisition scan speed by a factor of 200 (37). Helium
pressure was reduced to 5.0 x 10° torr (uncorrected) and a resonance ejection signal was
applied at 118,936 Hz with an amplitude of 2.4 V (peak-to-peak, endcap-to-endcap) to eject
m/Am = 33,200
80-
60- 1Ci3
0.039 u
40- 7 C13
204 |
m/z .
the ion trap. An average resolution of 12,560 was obtained. An average resolution of
12,210 was observed for the QITMS; thus the two modes of operation appear to be
analogous, as expected. The quadrupole was subsequently operated in mass-selected
fit" linear regression analysis of the mass resolution of m/z 502 calculated for each of the
modes of operation as a function of cooling time. The original and the regression data
show ~10% improvement in resolution obtained by adding the quadrupole and eliminating
the rf-isolation step.
are crucial to obtain optimal performance (39, 40). It is possible that the ion spatial and
velocity distributions at the exit aperture of the quadrupole mass filter provide better initial
injection conditions than those obtained using a lens injection system, accounting for the
generally improved performance of the hybrid over the QITMS. Data portrayed in Figure
4.7 indicate an enhancement in resolution when ions were not rf-isolated in the QITMS.
Nonlinear effects arising from the addition of higher-order fields to the quadrupole trapping
field may be a contributing factor to the improved performance of the Q/QITMS when rf-
isolation in the trap was not employed. One effect of higher-order fields is to create a shift
in the secular frequencies of ions in the ion trap (41). This frequency shifting could cause
dispersion of the ion packet during rf-isolation due to the excitation of some of the
43 as a function of cooling time. Cooling time was increased from 0 to 50 ms in 5 ms
steps and 30 QITMS microscans were summed and acquired. The high resolution mass
spectrum was obtained by slowing the QITMS acquisition scan speed by a factor of 100
(37). HB indicates mass-selected injection with no rf-isolation in the ion trap, ® indicates
oS Sp Ov Se O€ S@ OC ST OL
+ 000CT
increased (41). The linear decrease in resolution as a function of cooling time for all modes
fly" during sequential injection of ions into the ion trap. Cooks and Morand demonstrated
the use of the ion trap as both collision cell and mass analyzer by adjusting the energy
difference between an ion trap and a quadrupole mass filter (22, 23). In a similar
experiment, an ICL procedure was written to step the quadrupole from 50 u to 650 u in 10
u steps with a 10 u wide scan window. The value of the de voltage at which the ion trap
electrodes were floated was decremented each time the mass bin was reset to 50 u. As a
result, the relative injection energy of the ions from FC-43 was increased. Figure 4.8
portrays signal intensity for the resultant selected ion chromatograms plotted against the
trap float voltage. Every group of 60 QITMS scans represents one value of the float
voltage. The peaks labeled with circles in the ion chromatogram for m/z 264 show the
post-source fragmentation produced by the sequential injection of m/z 502 into the ion trap.
The more intense peaks in the chromatogram result from the sequential injection of m/z
264 into the mass spectrometer. The ion injection energy is a parameter that affects the
trapping efficiency (23). In this example, m/z 502 is trapped most efficiently at a float
voltage of -10 V while the optimal trapping voltage for m/z 264 is -12 V. Fragmentation
for m/z 502 is maximized at -11 V. This type of experiment may elucidate the trap float
trap float voltage increased the relative injection energy, leading to increased fragmentation
over a narrow voltage range. Shown are the selected ion chromatograms for m/z 502 and
m/z 264. Each peak corresponds to a different value of the float voltage. Lower intensity
peaks in the SIC for m/z 264 result from fragmentation of m/z 502, and higher intensity
40 7
4.119
40 |
4.552
singly-charged peptides produced by Cs* bombardment. The dissociation of these
molecules was then investigated using resonance excitation of the trapped ions.
induce fragmentation of selected ions by bringing the secular oscillation frequency of the
ion into resonance with the frequency of the auxiliary signal (24). Figure 4.9 compares the
fragmentation spectrum obtained using the ion trap with that observed on the Q/QITMS
when resonance excitation was applied to the molecular ion of human angiotensin I (MW
1296). Angiotensin I was co-deposited with a 1:1 glycerol:thioglycerol matrix onto the
probe tip and ions were generated by pulsed Cs” bombardment. The molecular ion was
isolated by a combination of forward and reverse scans during operation with the QITMS.
Utilizing the hybrid instrument, a 10 u wide window was centered around m/z 1296.
Precursor ions were filtered through the quadrupole mass filter and trapped and then a
product mass spectrum was generated by resonance excitation.
values for the sequence ions delineated. Nomenclature is according to that proposed by
Roepstorff and Fohlman (43). Observed ions are underlined. An asterisk indicates ions
detected using the hybrid but.not seen using the QITMS. The nomenclature b, ,+18 is
used to represent signal from ions produced via the release by cyclization of the C-terminal
amino acid followed by retention of the carboxyl group on the new C-terminus (44).
Shown in Figure 4.9(a) is the fragmentation mass spectrum obtained on the QITMS-only
instrument. The scale was expanded by a factor of 10 from m/z 400 to m/z 1170. Some
representative fragment ions are labeled. Enough signals were present from high mass b-
ring electrode was set to bring the ion to a q, value of 0.3 and a signal at 118,111 Hz and
1.44 V_ (peak-to-peak, endcap-to-endcap) was applied for 30 ms across the endcap
electrodes. All ions with m/z below 430 were ejected from the trap due to the amplitude of
the rf voltage applied to the ring electrode during the excitation period. Ions were cooled
for 3 ms before ejection from the ion trap. 14 microscans were summed to provide the
mass spectra. The expected peptide sequence masses are shown and observed signals are
underlined. An asterisk indicates ions observed utilizing the hybrid and not observed
utilizing the QITMS. Ions labeled b, +18 presumably result from loss of the C-terminal
amino acid with retention of the carboxyl group. (a) Ions were rf-isolated in the ion trap
for QITMS operation. (b) Mass-selected injection was performed by the quadrupole.
Asp Arg Val Tyr Ile His Pro Phe His Leu
1297 1182 1026 926 763 650 513 416 269 132 y-type
1184 1069 912 813 650 537 400 303 156 by-1+18
a) Pt . i
1005 x10 by || o/b +18
ION TRAP Nal |
807
_ (M+H)t
607 Pro
"(bgt 18)* /
be ay
40- Y Pals é \
¥4
1007 x10 Dy | b* 1
HYBRID ] Sa |(MEDT
Yo/b+18
80-4 ol
60-5.
J be+18)*
“0 befbgt18 be/ye (at!8)
, “39
bg
20-
400 600 800 1200
to surface interactions (35). The signal at m/z 1051 results from loss of ammonia or water
from the b,+18 ion.
by the quadrupole followed by resonance excitation in the ion trap. The two spectra have
many similar features including the number and types of sequence-specific fragment ions
observed. A striking difference between the two spectra is the signal-to-noise ratio, far
better in the hybrid instrument than when only the QITMS was utilized. In a typical
example, the S/N ratio for the b,+18 ion at m/z 912 was 12.3 for the hybrid compared to
5.1 for the QITMS. S/N ratios for m/z 1182/1184 were 1212 for the hybrid and 294 for
the QITMS. The quality of the mass spectrum was typically better using the Q/QITMS
than the ion trap, especially in the low molecular weight region of the spectrum where
relative ion abundances were small. Over the course of many experiments, S/N ratios
varied for both modes of operation but were typically 20%-30% higher for the hybrid than
the QITMS. The most probable explanation for these improvements in the signal quality is
the reduction of space-charge and chemical noise. Low-mass matrix ions typically are
present in great excess over analyte ions and quickly fill the trap to the space-charge limit.
The fundamental rf level during ion injection cannot be set to eject these ions without
compromising trapping efficiency for the ions of interest. The mass filtering action of the
quadrupole serves to reduce space-charge and chemical noise produced by unwanted ions
and improves the quality of the fragmentation mass spectrum for the isolated molecular ion
being studied. The improved resolution of the Q/QITMS also affords the observation of a |
series of b, +18 ions that are not clearly noticeable in the ion trap data, providing a mass
was investigated as a function of post-excitation cooling time in the QITMS for the various
modes of operation, analogous to experiments performed by March et al. on n-
butylbenzene (45). Cooling time was increased in 1 ms steps from 0 - 15 ms, then in 5
ms steps from 15 - 60 ms. Approximately 12 microscans were acquired for the
unattenuated precursor ion, and 30 microscans were acquired for the MS/MS experiment.
Calculated peak areas for each microscan were summed and normalized by the number of
microscans acquired. The fragmentation efficiency was calculated by dividing the peak
area of the unattenuated precursor ion by the sum of the peak areas of the product ions.
Linear regression analysis was performed on the resultant data. Results of the regression
analysis exhibited similar trends to those observed for the high resolution cooling time
experiment shown in Figure 4.7. The performance, measured by the fragmentation
efficiency, was improved for the hybrid when compared with the QITMS. The percent
improvement in fragmentation efficiency obtained on the hybrid compared to the ion trap
was found to increase linearly as a function of cooling time (4% - 30%). Efficiency was
maximized when the precursor ion was not rf-isolated in the ion trap. The efficiency
difference between the non-rf-isolating mode and the rf-isolating mode of operation was
also found to increase linearly as a function of cooling time. Performance dropped overall
for long cooling times, unlike the results found by March et al. for small molecules (45).
Decreased efficiency for long cooling times may be attributed to ion loss due to the effects
capable of obtaining both molecular weight and structural information for mixtures of
molecules, has been assembled. Components from mixtures of gas phase ions were
sequentially injected into the ion trap and detected. Improvements in chromatographic
resolution of the ion current for a simple mixture of angiotensin II and [Val*]angiotensin II
were demonstrated by increasing the number of data points taken over a limited mass
range. This two-dimensional scan mode of operation will be useful for the analysis of
complex mixtures. Mass-selective injection was utilized to reduce space-charge induced
effects such as signal suppression and decreased resolution. Performance of the hybrid for
high-resolution and MS/MS experiments was typically better than that achieved by the
QITMS. Eliminating rf-isolation of ions in the ion trap provided the best results,
suggesting that step scanning the hybrid is an excellent method of pre-processing the ion
population for subsequent analysis. A shortcoming of the Q/QITMS is the bin scan time,
4.6 References
Phys. 67 285-294. |
(1981) Int. J. Mass Spectrom. Ion Phys. 39 125-132.
McLuckey, S. A. (1990) Int. J. Mass Spectrom. Ion Proc. 96 117-137.
Int. J. Mass Spectrom. Ion Phys. 30 127-136. |
Ion Proc. 77 49-61.
8. Glish, G. L., McLuckey, S. A., Ridley, T. Y., and Cooks, R. G. (1982) Int. J. Mass
Spectrom. Ion Phys. 41 157-177.
Spectrom. 2 142-145.
(Longevialle, P., Ed.), 11A, Elsevier, London, p. 286.
34th ASMS Conf. on Mass Spectrom. and Allied Topics, Cincinnatti, OH, American
Society for Mass Spectrometry, p. 799.
Allied Topics, Boston, MA, American Society for Mass Spectrometry, p.1.
(Wineforder, J.D.; Kolthoff, J.M., Eds.) in Chemical Analysis: A Series of Monographs
on Analytical Chemistry And Its Applications, V102, Wiley and Sons, New York, p. 266.
E. (1984) Proc. of the 32nd ASMS Conf. on Mass Spectrom. and Allied Topics, San
Antonio, TX, American Society for Mass Spectrometry, p. 513.
Spectrom. 3 62-66.
Mass Spectrom. Ion Proc. 98 209-224.
F. W., Ed.) Wiley and Sons, New York, Chapter 8.
Mass Spectrom. and Allied Topics, June 3-8, Tucson, AZ, American Society for Mass
Spectrometry, p. 1462.
Spectrom. and Allied Topics, June 3-8, Tucson, AZ, American Society for Mass
Spectrometry, p.1460.
Ion Proc. 105 13-29.
Todd, J. F. J. (1987) Anal. Chem. 59 1677-1685.
62 2162-2172.
59 449-454.
W. (1994) Anal. Chem. 66 313-318. |
Commun. Mass Spectrom. 7 20-26.
Commun. Mass Spectrom. 3 225-229. b) The source was floated at6 kV. Cesium ions
were extracted from the filament region with a grid lens held at -0.5 kV with respect to the
high voltage and were pulsed into the sample region utilizing a gating lens pulsing at -381
V (gate open) and +35 V (gate closed) with respect to the high voltage.
of the 36th ASMS Conf. on Mass Spectrom. and Allied Topics, June 5-10, San Francisco,
CA, American Society for Mass Spectrometry, p. 634.
Spectrom. Ion Proc. 103 1-19. b) The presence of adducts 11 mass units higher than the
(M+H)* ion, often observed with this instrument, may be indicative of surface interactions.
36. Williams, J. D., Cox, K. A., Cooks, R. G., Kaiser, R. E., Jr., and Schwartz, J. C.
(1991) Rapid Commun. Mass Spectrom. 3 327-329.
— 2198-204.
39, Reiser, H.-P., Julian, R. K., and Cooks, R. G. (1992) Int. J. Mass Spectrom. Ion
Proc. 121 49-63.
Proc. 123 86-96.
4 917-929.
Mass Spectrom. 21 226-241.
j 249-257.
45. Liere, P., Blasco, T., March, R. E., and Tabet, J.-C. (1994) Rapid Commun. Mass
Flowrate Electrospray lonization
peptides is described. An ion source was modified and interfaced to a hybrid mass
spectrometer that was described in Chapter Four. Phosphopeptides in a tryptic digest of o-
casein were identified using a novel method of scanning the hybrid. Two innovative
methods of applying voltage to the sample were developed. The first utilized a platinum
wire aligned coaxially with a fused silica transfer line and epoxied into a pulled glass
micropipette needle. This configuration on the hybrid provided detection of as little as 75
amol of a mixture of angiotensin I and melittin. A platinum wire was also inserted through
the sidewall of a piece of Teflon tubing to create a liquid junction. This configuration
facilitated connection of the microspray needle to capillary electrophoresis (CE) and HPLC
columns as well as to fused silica. One fmol of angiotensin was loaded onto a column and
on the order of 10 fmol loaded onto the membrane. The preliminary data indicate great
promise for the development of a sensitive and fast multi-dimensional chromatography
becoming the preferred analytical technique for biomedical and biochemical applications.
A recent review discusses the application of LC-MS to the analysis of drug metabolites,
compounds of pharmacological interest, peptides, proteins, glycoproteins, glycolipids, fatty
acids, vitamins, steroids, nucleic acids, drug conjugates, biosynthetic peptides, and
recombinant proteins (1). Electrospray ionization (2-5), with flowrates of 1-10 uL/min,
has emerged as the primary mode of ionization for the sensitive analysis of mixtures of
biomolecules. A series of innovations have decreased the limits of detection of the
technique to the femtomole to zeptomole level (6-9). High sensitivity has primarily been
achieved by lowering flowrates of effluent directed into the electrospray ionization source
by 1-2 orders of magnitude and reducing transfer line, column, and needle sizes (6, 8, 10,
11) in order to increase the effective sample concentration at the needle tip. This typically
involves extensively splitting the solvent flowrate of ~160 LL/min from an HPLC, usually
‘done pre-column. The extent of splitting depends upon the size of the column, with
smaller ID columns requiring nanoliter to microliter flowrates (6). A limitation of the
sensitivity separation of mixtures. The low flowrates provided by CE afford facile
coupling to microspray interfaces and exploit the sensitivity advantage of the technique. A
limitation of CE is that separation is poor for relatively complex mixtures. Although high
sensitivities are reported, injection of a concentrated sample is required due to the small
volumes that are typically injected onto the columri. Typical samples of biological interest
are generally quite dilute and not compatible with the requirements of CE. A recent
refinement provided by Naylor and co-workers is the use of a hydrophobic membrane to
concentrate dilute samples prior to injection onto a CE column (17, 18). Dilute solutions
(~300 amol/mL) were concentrated and eluted using 100% methanol onto a CE column,
affording detection of peptides at low attomole levels. In addition, the membranes were
useful for washing samples to remove salts and biological debris prior to CE-MS analysis.
electrospray process. A number of different methods have been employed to create an
electrical contact while minimizing the dead volume. CE capillaries and electrospray
needles have been coated with gold and connected to a metal surface bearing a potential (8,
12, 19). The process for capillaries involves etching the tips in hydrofluoric acid, coating
them with a silanizing solution, then sputter-coating them with gold. Tips produced using
this time-consuming process typically last for one day before the gold wears off (20).
and extensive optimization is required (21). Another widely used approach is the liquid
junction design presented by Henion and co-workers (13). A needle and a transfer line or
column are placed 10-20 um away from each other in a liquid reservoir that is charged.
Charged liquid flows into the needle while the small gap reduces the possibility of analyte
loss. The gap distance is crucial for optimization and is difficult to reproduce, providing a
limitation to this technique. A recent innovation by Smith and co-workers is the use of a
liquid junction provided by epoxying a length of microdialysis tubing to the needle and CE
column and placing the assembly in a reservoir provided by an Eppendorf pipette tip and
charged by a piece of copper wire (20). Changing buffer conditions within the reservoir
was used to shift the charge-state distribution of bovine carbonic anhydrase II and could be
useful in the study of non-covalent interactions. Finally, a metal union is employed to
connect the needle and column or transfer line, creating a liquid junction without the use of
a large reservoir (6, 11). Chelating interactions of peptides with the metal appears to be a
limitation of the technique. A platinum sheath tube inserted in the union and epoxied to the
source and its application to peptide mixture separations. New needle types and liquid
junctions are investigated and a robust, inexpensive, and easy-to-use liquid junction is
described. A unique method of mass spectrometric scanning is employed for the
identification of phosphopeptides in a mixture. Separation of peptides using step elutions
attached to a three-dimensional positioning device (Edmund Scientific Corp., Barrington,
NJ, USA). An aluminum plate was recessed into the sample stage and dc voltage (1-2
kV) was applied to the plate. A brass tab was used to anchor the microspray needle to the
plate and transfer voltage to the sample via the liquid junction. The configuration is
diagrammed in Figure 5.1. Sample was typically infused through a length of 365 um OD
x 50 um ID fused silica (Polymicro Technologies, Phoenix, AZ, USA) using a 10 UL
gas-tight syringe (Hamilton Corp., Reno, NV, USA) pushed by a programmable syringe
pump (Harvard Apparatus, South Natick, MA). Flowrates ranged from 50 - 200 nL/min
. interfaced to the hybrid mass spectrometer. A heated capillary (Finnigan MAT, San Jose,
CA, USA) was inserted with reverse geometry in place of a glass capillary in an
electrospray source (Analytica of Branford, Branford, CT, USA). The use ofa heated
system were modifications of an existing electrospray ionization source and were used on
BOTS Pasay
ysiH
A sud’] Axeyidesy d[P90N
sosua] gqny pojeoH
be inserted into the capillary itself and enhances the sensitivity of the technique. Control of
the capillary temperature was provided by a programmable temperature controller (Omega
Engineering, Inc., Stamford, CT, USA) interfaced to a linear 24 V 2.4 A de power supply
manufactured by Sola and purchased from Newark Electronics (Chicago, IL, USA).
Maximum temperature provided by the power supply was 175°C as displayed on the
controller LED. A unique tube lens configuration was used to focus ions onto the
skimmer and transfer heat from the heated capillary to avoid melting the vespel insulating
sleeve. The skimmer and lensing system of the Analytica source were left unmodified and
served to further decluster ions and focus them into the acceptance aperture of the
quadrupole mass filter. The capillary temperature was typically set to 135°C and the
capillary voltage was 15.8 V, with the skimmer held at ground. The tube lens voltage was
23.8 V. Voltages for the three element lensing system were as follows: Lens 1 1.1 V,
micropipettes (World Precision Instruments, Sarasota, FL, USA) held under tension until
they pulled into a needle shape using a PUL-1 puller (World Precision Instruments). The
needles were opened by gently touching the tip to the lab bench. Polyimide coating was
burned off of the end of a short piece of fused silica transfer line (365-500 um OD x 50
uum ID). The needle tips were trimmed to a length of ~1 cm and the transfer line was
inserted into the tip and epoxied to the glass (EpoTek, Billerica) MA, USA). Liquid
junctions were created by inserting a 1.5 cm length of 125 um diameter platinum wire
(Scientific Instrument Services, Inc., Ringoes, NY, USA) coaxially with the transfer line
binder clip) was attached to a piece of 365 tm OD x 50 um ID fused silica taped to the lab
bench. The smallest blue flame from a microtorch (Scientific Instrument Services, Inc.)
was applied until the capillary began to neck down, then was withdrawn as the fused silica
the microscope to open them. Needle tips used for CE experiments were trimmed so as to
keep the ID of the capillary approximately constant while the OD was decreased. Needles
glass micropipette needle with a fused silica transfer line and a platinum wire inserted
coaxially and epoxied. (b) A metal union, generally stainless steel tubing epoxied to a
fused silica transfer line and a pulled capillary needle. Alternatively, Valco unions with
HPLC fittings were used. (c) A novel junction made of Teflon tubing and platinum wire
with a fused silica transfer line and a pulled capillary needle or a glass micropipette/fused
Micropipette Fused Silica
wo OS
Connected to HV
Transfer Line
Connected to HV
Necale Capillary Tubing Transfer Line
Connected to HV
together using a metal union as shown in Figure 5.2(b). Typically, 1.5 cm long pieces of
22 Ga stainless steel tubing were used as the union. Needles were inserted through one
end and a transfer line was inserted through the other end until the two ends touched. The
fused silica was then epoxied to each end of the tubing. Alternatively, commercially
available metal unions were investigated. Fused silica lines and needles were placed in
Peek tubing sleeves and butt-connected on either side of a zero dead volume stainless steel
diameter platinum wire inserted through the sidewall of a ~1 cm long piece of Teflon
tubing (250 um ID x 1.59 mm OD, Upchurch Scientific, WA, USA), through the core
and into the other sidewall. Needles and transfer lines were pushed into the ends of the
tubing and the Teflon formed a seal about the capillaries. The configuration is shown in
Figure 5.2(c). Both pulled micropipette and pulled capillary needles were used with this
type of junction. The distance between the fused silica capillaries ranged from 0.6 to 2
instrument described in Chapter 4 was used to develop the microspray source and
investigate the utility of unique mass spectrometric scans available with the quadrupole/ion
trap combination for identification of phosphopeptides (discussed below). The quadrupole
offset was typically set to -12.8 V and the resolution of the quadrupole set to 50-60% of the
maximum attainable value. The electrode assembly was floated at -4.2 V and ions were
gated into the trap using a tube lens pulsing from + 200V (gate closed) to -190 V (gate
open). The conversion dynode was operated at -10 kV with the electron multiplier set to -
was employed for the CE and chromatography experiments. The instrument configuration
is shown diagramatically in Figure 5.3. A voltage of 23.5 V was applied to the heated
capillary and the temperature was set to 200°C. Two rf-only octopoles were used to
transmit ions into the ion trap. The offset of the first octopole was -3.2 V, and that of the
second octopole was -6.2 V. Both had an rf voltage amplitude of 400 V,, applied to the
rods. The inter-octopole lens voltage was -26'V. The electrode assembly was floated at a
dc offset of -10V and ions were gated into the trap using a tube lens gating from 55 V (gate
closed) to -200 V (gate open). Ions were detected using a conversion dynode with the
electron multiplier set to -1 kV. Automatic gain control (AGC) was used to maintain a
/spoukg WoIsI9AU0,) sua’] 2[0d0}90-197U] JOULES
| /suayy aqny,
Thorsteinsdottir and co-workers (24) modified as described by Figeys et al. (9). Pressure
loading was applied to flow reagents through a 66 cm length of 365 um OD x 50 um ID
fused silica capillary. Table 5.1 details reagents and reaction times for the preparation. A
high voltage supply (Glassman High Voltage, Inc., Whitehouse Station, NJ, USA) with a
platinum ribbon (Scientific Instrument Services) soldered to the electrode was used to
deliver 25 kV to buffer or sample solutions placed in a microcentrifuge tube inside of a
lucite chamber. The CE column was inserted through a Teflon fingertight fitting and was
threaded through a holder to ensure the capillary and electrode would stay within the
confines of the microcentrifuge tube. At 25 kV applied voltage, the flowrate was measured
USA) was opened on the ends using a needle and bored out with a 325 um diameter piece
Time (hr) Pressure
(psi)
0.5 M NaOH 0.7 500
H,O 0.2 500
3M HCl 2.5 300
110°C with He 20 40
10% (v/v) 3-aminopropyltrimethoxysilane in 3 100
dry toluene
102°C with He 19 100
dry toluene 0.4 100
Extraction Disks with SDB-XC, Lot #710009, 3M, St. Paul, MN, USA) were obtained
from Varian (Harbor City, CA, USA) and were prepared in a manner analogous to that
presented by Naylor et al. (17). A short piece of fused silica (430 um OD x 320 um ID)
_ was employed to punch through the membrane disk. The end containing membrane
material was placed in the Teflon cartridge. Another piece of fused silica (217 um OD x
16 um ID) was used to push the membrane slug into the Teflon cartridge. Fused silica
of methanol followed by 30 wL of water with 0.5% acetic acid (v/v). Sample was diluted
in water with 0.5% acetic acid and 0.5 - 10 pL loaded onto the membrane cartridge. The
cartridge was subsequently washed with 10 WL water with 0.5% acetic acid to wash the
sample from the walls of the capillary onto the membrane. A short length of fused silica
was placed in the Teflon liquid junction and the membrane cartridge placed between the
short transfer line on one end and a longer transfer line on the other end that was connected
to the syringe and pump. The membrane cartridge was taken off-line and a Teflon
connector was used to clean the transfer lines and needles and to fill the lines with elution
melittin (MW 2847 Da, Cat. No. M-2272 ) and bovine o&—casein (MW 25 kDa, Cat. No.
V511A, Lot No. 5903801) was purchased from Promega (Madison, WI, USA) and
diluted in water to a concentration of 1 Ug/uL. Trypsin digestion was performed by
diluting ~20 nmol of solid o—casein in 30 UL of 50 mM ammonium bicarbonate, pH 8.
Trypsin (8 1g) was added to provide an enzyme to substrate ratio of ~ 1/60 and the
mixture was heated at 37°C for 18 hr. The reaction was stopped by adding 5 wL of glacial
acetic acid. Sample was subsequently concentrated using a Speed-Vac (Savant
Instruments, Farmingdale, NY, USA) and diluted in water with 0.5% acetic acid to a final
concentration of ~377 pmol/L. The stock solution was stored at -20°C. One wL of stock
solution was diluted in 377 uwL of 0.5% acetic acid to provide the sample. Dilutions from a
1 nmol/uL stock solution of angiotensin I and melittin were similarly prepared to provide a
1 pmol/uL sample in 0.5% acetic acid. Angiotensin and melittin were mixed together to
provide an equimolar sample. For high sensitivity experiments, samples were diluted
from 1 pmol/L to 100 fmol/uL or 1 fmol/uL in a microcentrifuge tube. Samples for
and to use this source to investigate methods of mixture analysis. Results for the
performance of the source are presented for selected peptides. A tryptic digest of o—casein
either small bore (~6 [um ID) fused silica capillary with an etched tip (6, 10) or fused silica
capillary (20-50 um ID) pulled to a small exit diameter (~2-3 um ID) (8, 11). The second
type of needle developed by Wilm et al. utilizes a gold sputter-coated pulled glass
micropipette as a needle (~2-3 um ID exit diameter) (19, 25). The coating apparatus is
expensive and the coating does not last long on the needles, even when the glass is
derivatized (8, 20). Needle lifetimes are on the order of 3 hrs. Lee and co-workers have
reported on the performance of 350 um OD x 150 tm ID fused silica capillaries pulled to
aneedle with 150 uum OD x 25 tm ID transfer lines placed inside to minimize the dead
volume (11). When a PVDF frit is placed in-line, needle lifetimes are extended
significantly to 8-12 hr. Combining these methods, we have inserted a transfer line into a
pulled glass micropipette needle as described above. Rather than gold coating, voltage was
supplied to the liquid sample either through the use of a stainless steel union epoxied to the
needle or via a platinum wire inserted into the needle or into a Teflon liquid junction.
in Figure 5.5. Serial dilutions of an equimolar mixture of angiotensin I and melittin were
used to investigate detection limits on the hybrid quadrupole mass filter/quadrupole ion trap
mass spectrometer. A pulled glass needle with an epoxied platinum wire was employed as -
the liquid junction. A 50 fmol/uL solution was infused into the source at a rate of 50
melittin. The quadrupole mass filter was used in rf-only mode to transmit all ions into the
ion trap. The ion gate was opened for 50 msec and all ions above 55 u were stably trapped.
Ions were cooled in the trap for 10 msec prior to ejection by ramping the amplitude of the
fundamental rf from 450 - 7500 V,,, with an auxiliary signal at 119,936 Hz and 8.8 Vv
(peak-to-peak, endcap-to-endcap) placed on the endcap electrodes to enable resonance
ejection and mass range extension. The mass spectrum is the result of summing 3
+4
711.7
100
807
607
melittin
+3
949.3
melittin |
+5
569.4
404
angiotensin I
204 “+3
433.2
i Va vl ul
- plz
material. The signal-to-noise ratio for the quadruply-charged ion from melittin (m/z 712)
was calculated at 8:1. Calculated detection limits were determined to be in the 5-10 amol
and the pulled capillary needles revealed that the micropipette needles lasted longer without
clogging and provided a higher signal-to-noise ratio (although total ion current was
generally slightly lower) than did the capillary needles (data not shown). The performance
of the needle when the platinum wire was inserted was compromised by depolymerization
of the epoxy due to the application of the high voltage. This caused the epoxy to cover the
platinum wire and prevented the application of voltage to the liquid in the needle tip. The
micropipette needles also required steady solvent flow. Air bubbles in the transfer line
caused significant instabilities in the spray. This was a particular problem when the needles
liquid sample for electrospray ionization. Sheath flow sources and reservoir-type liquid
junctions are tedious to optimize and result in dilution of the analyte. Metal unions utilizing
HPLC fittings can be difficult to troubleshoot at low flowrates because leaks are difficult to
find. In addition, changing needles generally requires new ferrules and sleeve fittings and
and/or solvent interactions effect the integrity of the epoxy and lead to an increased
background of epoxy peaks. Finally, gold-coated needles have been employed to transfer
voltage to a sample. The needles are held at ground and a potential is placed on a glass
capillary ~1 mm away. Needles typically last for up to 3 hours (19). When heated
capillaries are used, voltage is generally placed on the needle. The gold coating is easily
removed when placed either directly in contact with an electrode or close enough to an
platinum wire pushed through the sidewall of a small length of Teflon tubing, described
above. The junction appears to be versatile and robust. Peptides do not interact with the
inert material, thus interaction effects are eliminated. Needles and transfer lines can be
inserted and removed with ease, facilitating troubleshooting. Removal of epoxy from the
system eliminates many of the background signals. Both pulled glass micropipette needles
with an epoxied transfer line and pulled capillary needles can be used. Some epoxy
background was observed with the micropipette needles; however, it was of low
abundance and was virtually eliminated upon optimization of spray conditions (data not
shown). Performance of the junctions as measured by spray stability and signal-to-noise
of the sample appeared to be equivalent to performance obtained using standard metal
molecules. Here we describe a number of approaches used to extract information from
spectrometers (26). The first quadrupole transmits a given m/z, collisions occur in. the
second quadrupole, then the third quadrupole transmits only those ions at a given m/z
below the precursor ion transmitted by the first quadrupole. For example, when looking
for losses of phosphoric acid from a doubly-charged phosphopeptide, the transmission m/z
of the third quadrupole is offset by m/z 49 from the transmission m/z of the first
quadrupole. This type of experiment cannot easily be done using an ion trap mass
spectrometer (27). The hybrid mass spectrometer, described in the preceeding chapter,
was employed to develop a new type of neutral loss scan designed to identify the presence
epoxied into the tip (Figure 5.2(a)) was used to ionize a tryptic digest of &-—casein at a
concentration of 3.7 pmol/uL flowing at 100 nL/min. Two peptides from the mixture
MS/MS was performed in the ion trap and only the product ions falling within a 60 u mass
window below the precursor mass were ejected detected. For example, when analyzing
m/z 831, only product ions with m/z values between m/z 750 and m/z 810 were ejected
from the ion trap and detected. Resultant mass spectra are shown in Figure 5.6. Figure
5.6(a) provides the mass spectrum obtained from analysis of product ions with m/z values
between m/z 615 and m/z 675. No signal is present in the spectrum indicating no loss of
49 u from m/z 693. Conversely, the peak shown in the mass spectrum in Figure 5.6(b)
shows clearly that m/z 831 corresponds to a doubly-charged phosphorylated peptide since
molecules. Separations can be performed in a shorter time than is possible by liquid
chromatographic means and often with higher resolution. Small sample requirements and
low flowrates inherent in CE make the technique ideal for interfacing to microspray
microspray source using the Teflon liquid junction and a pulled capillary needle. The LCQ
ion trap was employed for this experiment. The software on the newly commercialized
allowed into the ion trap for 100 msec. Ions were cooled for 10 msec then ejected using a
supplementary signal at 119,936 Hz with an amplitude of 8.8 V (peak-to-peak, endcap-to-
endcap). All ions above m/z 55 were trapped. (a) The quadrupole injected m/z 692-694
into the trap. MS/MS was performed using a q, value of 0.315, offset by ~5% from the
theoretical g, value of 0.3. The resonance excitation signal was at 118,811 Hz with an
amplitude of 24.8 V (peak-to-peak, endcap-to-endcap). The amplitude of the fundamental
rf was ramped from 2365 V,,, to 2596 V,,, at a resonance frequency chosen to triple the
mass range, affording detection of ions between m/z 615 and m/z 675. The expected
neutral loss from a doubly charged phosphopeptide would appear at m/z 644. (b) The
quadrupole injected m/z 830-832 into the trap. MS/MS was performed using a q, value of
0.315, offset by ~5% from the theoretical g, value of 0.3. The resonance excitation signal
was at 118,811 Hz with an amplitude of 28.8 V (peak-to-peak, endcap-to-endcap). The
amplitude of the fundamental rf was ramped from 2885 V,, to 3115 V,, at a resonance
frequency chosen to triple the mass range, affording detection of ions between m/z 750 and
m/z 810. The expected neutral loss from a doubly charged phosphopeptide appears at m/z
O8L — | O9L
OcL QL9 099 0S9 Ovo 09 _0¢9 O19
(q
casein. The top panel of Figure 5.7(b) shows the unit resolution full mass-range spectrum
of the entire digest. MS/MS on the ion with m/z 880 provided the mass spectrum shown
in the second panel. Ions below 245 u were ejected upon the application of the resonance
excitation pulse. A third stage of mass spectrometry, depicted in the third panel, provides
additional low mass sequence ions while the sequence is completed using a fourth stage of
mass spectrometry. The amino acid sequence was deduced to be
HQGLPQEVLNENLLR. The experiment took approximately 10 min to perform on the
LCQ whereas manually tuning all of the parameters would have taken at least 3 hrs on the
ITMS ion trap. The following experiments were executed on the LCQ due to the ease of
of fused silica capillaries as described above to perform CE experiments. The sensitivity
of the technique was investigated using serial dilutions of angiotensin I. A 1 pmol/uwL
solution of angiotensin I in 0.1% acetic acid was diluted to 50 fmol/uL and injected onto
the column using a voltage of -25 kV applied for 6 sec, resulting in the injection of 1 fmol
of material onto the column. The column was then placed in buffer solution (10:90
methanol:0.1% acetic acid) and sample was eluted using ‘an applied voltage of -25 kV
coupled to a microspray voltage of 1.7 kV. The resultant mass spectrum is depicted in
Figure 5.8 with the selected ion chromatogram displayed as an inset. The peptide eluted
from the column over ~20 sec time period. Strong signal is observed from the triply-
charged and doubly-charged ions with excellent signal-to-noise. Peaks at m/z 443, 523,
protein o-casein. A 500 fmol/uL sample solution was infused into a microspray
ionization source at a flowrate of 200 nL/min. The ionization time was automatically set
using automated gain control for the first three stages of mass spectrometry. The AGC
was disabled for the fourth stage and the ionization time was set to 400 msec to
compensate for the loss in sensitivity due to the performance of multiple stages of mass
spectrometry. Ten scans were summed for the MS, MS’, and MS? experiments. Fifteen
scans were summed for the MS‘ experiment. (a) Mass assignments for sequence ions
corresponding to MS? of m/z 880, MS? of m/z 436, and MS* of m/z 266. Observed
sequence ions are underlined. (b) The precursor ion at m/z 880 displayed in the top panel
was chosen for fragmentation. The b,"’ fragment ion at m/z 436, shown in the second
panel, was chosen for a further stage of fragmentation and the resulting mass spectrum is
exhibited in the third panel. The b,"’ fragment ion at m/z 266 was chosen to obtain the very
EQ 4
om | | Aa
xl aLaLs| a] s| 7] 5| 31 91412] 9/21 8
[+ Migd KOE A x 4w@& OT &é e
Yu
Ig 43
I+ 2
El
I+ A tlq--—- 4
I+ Wik
we ATS
ot
pclq---4 =
Hg--—. —
+ We
ol k-*4
ATR
Smeal
Og--—-. 4
i+ 4 “2
4A
1q-<— 48
z(H7+W) =.
yh
a r)
alk =i)
+4 ox 4°
[+
9q --—
J+ q SK
ro)
\O
2 ia
= ” :
+4 eK 2
Wh 4S
SZ fq----]
co eS
@—O 7 4--
T UJ J 1 t 5.
eooeoe9o 8
S506 FA
and
7 1H
i) en
¥,
Ite ®
14>
144° 9-~-
14-4
144
my
oS \O
(oe) fom)
[oe] wt
> oO 0 80
S [oe] io) wt N
Soul
by
m/Z
fmol of angiotensin I onto a positively-charged column followed by CE elution at -25 kV.
rT
551.5
878.4
579.4 |
703.3 821.8 964.3
‘| | ;
~2 times greater than is typically observed for CE of peptides. Subsequent use of a
commercially available column provided smaller elution bands and stronger signal for the
same experiment. A mixture of peptides generated by trypsin digestion of o-casein were
only partially separated using this method (data not shown). Separation of the mixture has
not been attempted on a commercial column; however, optimization should improve the
Biological samples are typically quite dilute; therefore, hydrophobic pre-concentration
membranes have been used extensively by Naylor et al. to concentrate samples prior to
injection onto CE columns (17, 18). A second limitation of CE is that the technique is
most successful in separating relatively simple mixtures of peptides. Biological samples
are typically composed of complex mixtures of molecules; thus it would be useful to
develop a concentrating and simplifying procedure to prepare samples for subsequent
surfaces by passing an organic solvent over the surface. The solvent and peptides compete
concentration membranes has involved elution of all peptides from the membrane with an
elution buffer containing 80% methanol and 20% water with acetic acid (17, 18). We
investigated the sensitivity of eluting peptides from the membrane interfaced to the Teflon
junction with a pulled capillary needle by binding a series of dilutions of angiotensin I to
the membrane and eluting using an 80% methanol elution buffer at a flowrate of 200
nL/min. The limit of detection was determined to be 10 fmol of sample loaded onto the
membrane from a 1 fmol/uL solution. The corresponding mass spectrum is exhibited in
Figure 5.9. The signal-to-noise ratio of the triply-charged ion was calculated to be 6:1 and
the elution peak width was ~ 1 min. One minute peak widths correspond to.passing one
column volume of elution buffer through the membrane. Sample loadings of 100 fmol
and 500 fmol of a 100 fmol/uL solution provided signal-to-noise ratios of ~20:1 (data not
shown). Graphical representation of the results is shown in Figure 5.10. The best fit to the
data was provided by a logarithmic function. In general, S/N ratios decrease in a linear
fashion; thus we assume that there are sample losses at low sample concentration causing
the apparent logarithmic relationship. The hydrophobicity of | angiotensin I can enhance
losses to sample tube walls, syringes, and transfer lines; thus a more hydrophilic peptide
fmol. Signal was observed at the single fmol level from loading 1 pL of a 1 fmol/uL
solution; however, there were many background ions of equal intensity. Low abundance
signal was also observed for 500 amol loaded onto the membrane from 0.5 uL of a 1
fmol/uL solution loaded onto the membrane and eluted using 80:20:0.5
methanol:water:acetic acid (v/v/v). The LCQ ion trap was scanned from 400 u to 1850 u
433.6 649.7
Tc
879.4
5415 o77.4 1118.2
hydrophobic membrane. The relationship appears to be logarithmic, suggesting the
OOT
and that further optimization of sample handling should further decrease the limit of
membrane cartridge. The bubbles disrupt the microspray, and pressure must be put on the
syringe pump to re-establish the spray. Spray is easily re-established using the pulled
capillary needles, but it is much more difficult to re-establish microspray using the
micropipette needles. More work must be done to determine how air bubbles can be
shown). The performance of the column was poor and ion signals from different peptides
were not well resolved because the -25 kV applied to the column removed
aminopropyltrimethoxysilane from the column walls, preventing efficient separation of the
peptides. Additionally, CE is typically most effective at separating peptides in relatively
simple mixtures and the column length was too short to effectively resolve the complex
mixture investigated. The use of hydrophobic membranes to simplify peptide mixtures
digest was loaded onto the membrane as described above. Peptides were eluted
isocratically at 30%, 50%, and 70% methanol in 0.5% acetic acid. Figure 5.11 illustrates
identified are denoted by "T.". A "P" in the subscript indicates the peptide was
phosphorylated. Peptides labeled with a "C" arise from cleavage due to chymotryptic
activity. Peptides from both types of casein were identified. High resolution scans were
KVNELSKDIG SESTEDQAME DIKQMEAESI SSSEEIVPNS VEQKHIQKED
VPSERYLGYL EQLLRLKKYK VPQLEIVPNS AEERLHSMKE GIHAQQKEPM
t i
IGSENSEKTT MPLW
T 67 | '
CSTFCKEVVR NANEEEYSIG SSSEESAEVA TEEVKITVDD KHYQKALNEI
Ty I 7 Ty,
acids from casein type 1 were identified and 16% of the amino acids from casein
type 2 were identified. One phosphopeptide at m/z 831 was observed. Casein is
known to contain five sites of phosphorylation which have been observed on a
triple quadrupole mass spectrometer. We have not yet determined why only one
observed and the data is summarized in Table 5.2. Only two ions, m/z 588 and m/z
693, appeared in more than one elution. Ions also tended to elute differentially
during the course of each elution. An example of the phenomenon is displayed in
Table 5.3 where the relative abundances of dominant ions change over elution time.
Scans occurring during 30 sec intervals were summed, providing the relative
abundances of the ions. Step elution coupled to CE where elution times are ~1-2
min could also provide another dimension of separation. Several CE analyses
analysis of peptides. A novel needle configuration composed of a fused silica
transfer line epoxied into a pulled glass micropipette needle tip was tested and
provided high sensitivity under infusion conditions. A unique Teflon/platinum
elutions of a casein digest peptide mixture from a hydrophobic membrane.
30 491, 588, 599, 685, 690, 765, 881, 912
50 588, 634, 693, 831, 886, 1010, 1023, 1064, 1267
acid.
Int.” 634 693 748 831 881 980 1267 1384
1 20.3 0 15.72 32.73 100 0 14.49 0
5 _ 100 85.34 53.89 22.31 25.95 47.91 79.16 45.38
6 66.88 100 28.12 24.79 25.07 81.92 63.26 71.69
7 31.84 100 0 15.46 0 55.22 25.92 56.67 |
8 24.41 100 0 16.6 16.4 62.9 21.37, 70.19
9 23.29 100 0 15.87 13.42 49.4 18.97 38.83
10 18.67 100 0 15.88 12.66 64.41 20.4 98.78
11 13.07 73.63 7.36 9.91 11.68 45.39 17.3 100
12 5.5 55.31 0 5.71 5.17 34.67 12.64 100
13 6.0 53.52 0 0 5.81 33.85 11.62 100
14 11.61 68.12 0 7.94 9.6 43.48 14.93 100
15 9.86 100 0 10.05 0O 55.13 8.04 78.66
16 0 100 0 0 0 65.65 9.56 90.4
scans during this interval were summed, providing the relative abundances shown above.
epoxy. The junction gave comparable performance to more traditional liquid junctions and
was significantly easier to use. The dead volume in the junction did not significantly
impair CE performance as determined by elution peak widths. LC performance using the
means of identifying given components of a mixture; however, this useful technique
cannot be implemented easily on a quadrupole ion trap. A neutral loss scan was developed
and successfully applied on a hybrid quadrupole mass filter/quadrupole ion trap mass
fo CE applications. Sensitivity at the 10 fmol level was demonstrated using step elution at
low flowrates. The membranes can be used as chromatographic devices in addition to pre-
concentration devices. Crude separation of peptide mixtures was accomplished by step
eluting peptides at different concentrations of organic. In addition, differential release of
peptides during the course of an elution can provide the opportunity of further mixture
refinement by performing multiple CE analyses during one elution. Other types of
10 361-368.
Science 246 64-71.
37-70.
613. |
Spectrom. 5 867-869.
W. (1995) Anal. Chem. 67 3802-3805.
68 1822-1828.
1021.
Chem. 59 1230-1232.
458 313-321.
Chem. 60 436-441.
Chromatogr. 554 311-327. |
584A.
19. Wilm, M., and Mann, M. (1996) Anal. Chem. 68 1-8.
Spectrom. 10 1175-1178.
(1994) Anal. Chem. 66 4450-4458.
Spectrom. 4 81-87.
480 339-349.
180.
26. Fetterolf, D. D., and Yost, R. A. (1983) Mass Spectrom. Rev. 2 1-45.
analysis of molecules of biological origin using a quadrupole ion trap mass spectrometer.
Of particular interest was the development of a sensitive methodology with which to
analyze peptide mixtures. An "off-line" technique utilizing an HPLC to separate peptide
mixtures resulting from enzymatic digests followed by MALDI-ITMS analysis was
successfully applied to locate sites of post-translational modification in the P protein from
Sendai virus. A hybrid quadrupole mass filter/quadrupole ion trap mass spectrometer was
constructed to investigate mixtures by transmitting one value of m/z at a time for analysis
in an ion trap. Finally, an "on-line" microspray ionization source coupled to a hydrophobic
membrane and a capillary electrophoresis interface was applied to explore the utility of
peptide mixture separation by high sensitivity multi-dimensional chromatography. A
description of these approaches and the results have been discussed in the preceeding
MALDI/FTMS instrument (2) with mass resolutions in excess of 10° (3, 4). FTMS has
also been employed to detect singly-charged biomolecules with molecular weights up to
157,000 u (5S). Emphasis in MALDI/quadrupole ion traps has been placed on improving
trapping efficiency, fragmentation efficiency, and ejection efficiency for MALDI-generated .
peptide ions (6-8). An improved external injection instrument has been utilized to analyze
peptide mixtures at high femtomole levels and may serve as the prototype for a
Spectrometer
quality of the mass spectra and in resolution and fragmentation efficiency. Slow scan
times due to the data porting time limited the utility of the approach for continuous
ionization techniques. The limitation could be ameliorated by utilizing only the TSQ
electronics to analyze and display the signal from the electron multiplier, thus eliminating
the need to transfer the data from the ion trap’s data system and reducing the bin scan time
of the quadrupole mass filter. Similar results could be obtained using tailored waveforms
to selectively inject sequential values of m/z into an ion trap. The new LCQ ion trap from
6.3 Mixture Separation by Low Flowrate Electrospray Ionization
source with unique needle and liquid junction configurations. The source was separately .
interfaced to capillary electrophoresis and membrane chromatography columns. The low
femtomole sensitivity obtained is sufficient for many biological applications and
optimization of conditions should further decrease the limit of detection of the technique.
Future experiments will entail coupling the hydrophobic membrane cartridge to the CE
column to perform two-dimensional chromatography. In addition, ion exchange
membranes will be used to obtain a further degree of mixture separation. The combination
of pre-concentration of dilute samples, CE, microspray ionization, and quadrupole ion trap
mass spectrometry should provide a sensitive, high throughput technique for the
simplification and analysis of complex mixtures of peptides generated from the study of
6.4 References
2. Li, Y., and Mclver, R. T., Jr. (1994) Rapid Commun. Mass Spectrom. 8 743-749.
3. Li, Y., McIver, R. T., Jr., and Hunter, R. L. (1994) Anal. Chem. 66 2077-2083.
5. Solouki, T., Gillig, K. J., and Russell, D. H. (1994) Anal. Chem. 66 1583-1587.
7. Doroshenko, V. M., and Cotter, R. J. (1995) Anal. Chem. 67 2180-2187.
Vita
Bobbi Furer. She received her bachelor of science degree in Engineering Physics from the
University of Colorado in June, 1989, and graduated with Special Honors. In 1987 she
worked as a summer intern at Argonne National Laboratory where she met Peter L.
Jonscher. They were married in 1991. Other summer internships were at Martin Marietta
Denver Aerospace Mission Operations (1988) and with Drs. Donald Burnett and Thomas
Tombrello at the California Institute of Technology (1989). In 1990 Karen joined the
research group of Dr. Leroy E. Hood at the California Institute of Technology and worked
in the mass spectrometry laboratory under Dr. John R. Yates II. She received a master of
science degree in Applied Physics from the California Institute of Technology in June,
1991. Spencer Lucas was born in April of 1992 and shortly thereafter Karen moved to
Seattle, Washington, to continue her research with Dr. Hood. In June of 1994 Raleigh
Lyle was born. Karen has accepted a post-doctoral position with Dr. Robert C. Murphy at
the National Jewish Center for Immunology and Respiratory Medicine in Denver,