1. The Future of Time-of-flight Mass Spectrometry
Marvin Vestal
Applied Biosystems
Framingham, MA

2. A Brief History of Time (of flight) with apologies to Stephen Hawking
1946 W.E Stephens, Phys. Rev. 69,641
“Advances in electronics seem to make practical a type of mass spectrometer in which microsecond pulses of ions are selected every millisecond from an ordinary low-voltage ion source. In travelling down the vacuum tube, ions of different M/e have different velocities and consequently separate into groups spread out in space. … This type of mass spectrometer should offer many advantages over present types. The response time should be limited only by the repetition rate (milliseconds)… Magnets and stabilization equipment would be eliminated. Resolution would not be limited by smallness of slits or alignment. Such a mass spectrometer should be well suited for composition control, rapid analysis, and portable use.”

3. Brief History of Time (of flight)
1948 Cameron & Eggers, Rev. Sci. Instr. 19, 605.
First working TOF
1953 Wiley & McLaren, Rev. Sci. Instr. 26, 1150
First practical TOF. Energy & Time Lag Focusing.
1959 Gohlke, Anal. Chem. 31, 535
GC-TOF
1963 Vestal & Wharhaftig, ASMS, 358.
Coincidence TOF, first ion counting TDC
1973 Mamyrin et al, Sov. Phys. JETP 37, 45.
Reflectron, higher resolution
1974 Macfarlane et al, Biochem. Biophys. Res. Comm. 60, 616
252Cf Plasma desorption. Proteins Fly!!!!

4. Brief History of Time (of flight)
1988 Karas & Hillenkamp, Anal. Chem. 60, 2299.
MALDI - Really big proteins fly!!!
1991 Dodenov et al, 12th Int. MS Conf.
O-TOF - Electrospray works with TOF
1993 Kaufman, Spengler & Lutzenkirchen, RCM 7, 902.
Post-source dcay MALDI
1994 Brown & Lennon, Sunriver, p63.
Delayed extraction MALDI - Makes MALDI-TOF routine
1996 Morris et al, RCM 10, 889.
Q-TOF
2001
TOF-TOF

5. Today’s Instruments Ionization Techniques TOF Analyzers
Electrospray o-TOF
Qq-o-TOF
MALDI linear TOF
reflector TOF
TOF-TOF
Trap-TOF

6. Why TOF? Speed Efficiency (Sensitivity) Dynamic Range Resolving Power
Mass Accuracy
Mass Range
Simplicity
Competitive in
All Respects
with
Unmatched
Speed

7. Speed Matters! Particularly with MALDI
More Samples - Higher Throughput
More Measurements/Sample
Better data - Precision of mass and intensity
Increased dynamic range

8. MALDI-TOF yesterday, today, and tomorrow
then now future
Laser Rate (hz) ,000
Acq. Time/Spect.(sec)
Spectra/day ,000 1,000,000*
*If we can process and interpret the results
Applications
Better sample utilization (>100,000 shots/spot)
Interface with separations
Molecular scanner
Tissue Imaging
1 micron resolution
=10,000 pixels

9. 50 Laser shots in 0.25 sec
on 125 femtomoles of trypic digest

10. E Coli beta-galactosidase, MW 116,483.9
16/20 matched, MOWSE Score 3e14, 23% sequence coverage
MTMITDSLAV VLQRRDWENP GVTQLNRLAA HPPFASWRNS EEARTDRPSQ QLRSLNGEWR FAWFPAPEAV PESWLECDLP
EADTVVVPSN WQMHGYDAPI YTNVTYPITV NPPFVPTENP TGCYSLTFNV DESWLQEGQT RIIFDGVNSA FHLWCNGRWV
GYGQDSRLPS EFDLSAFLRA GENRLAVMVL RWSDGSYLED QDMWRMSGIF RDVSLLHKPT TQISDFHVAT RFNDDFSRAV
LEAEVQMCGE LRDYLRVTVS LWQGETQVAS GTAPFGGEII DERGGYADRV TLRLNVENPK LWSAEIPNLY RAVVELHTAD
GTLIEAEACD VGFREVRIEN GLLLLNGKPL LIRGVNRHEH HPLHGQVMDE QTMVQDILLM KQNNFNAVRC SHYPNHPLWY
TLCDRYGLYV VDEANIETHG MVPMNRLTDD PRWLPAMSER VTRMVQRDRN HPSVIIWSLG NESGHGANHD ALYRWIKSVD
PSRPVQYEGG GADTTATDII CPMYARVDED QPFAVPKWS IKKWLSLPGE TRPLILCEYA HAMGNSLGGF AKYWQAFRQY
PRLWGGFVWD WVDQSLIKYD ENGNPWSAYG GDFGDTPNDR QFCMNGLVFA DRTPHPALTE AKHQQQFFQF RLSGQTIEVT
SEYLFRHSDN ELLHWMVALD GPKPLASGEVP LDVAPQGKQL IELPELPQPE SAGQLWLTVR VVQPNATAWS EAGHISAWQQ
WRLAENLSVT LPAASHAIPH LTTSEMDFCI ELGNKRWQFN RQSGFLSQMW IGDKKQLLTP LRDQFTRAPL DNDIGVSEAT
RIDPNAWVER WKAAGHYQAE AALLQCTADT LADAVLITTA HAWQHQGKTL FISRKTYRID GSGQMAITVD VEVASDTPPHP
ARIGLNCQLA QVAERVNWLG LGPQENYPDR LTAACFDRWD LPLSDMYTPY VFPSENGLRC GTRELNYGPH QWRGDFQFNI
SRYSQQQLME TSHRHLLHAE EGTWLNIDGF HMGIGGDDSW SPSVSAEFQL SAGRYHYQLV WCQK

11. 1000 Laser shots(5 seconds)

12. 10,000 laser shots, increased gain Low masses suppressed (50 seconds)

13. 10,000 laser shots Focused at low mass (50 seconds)
VTLR ?
EVR

14. Summary of Database Searching Results
1000 Laser Shots, 20 ppm window
m/z No. No Match Seq.Cov. Mean Error Data Tol.
S/N Sub. Match % % ppm ppm
Total

15. Peaks detected down to S/N =3 52/196 matched
M/z , 1000 laser shots
Peaks detected down to S/N =3
52/196 matched
Number of
Peptides
Matched
Error (ppm)

16. E Coli beta-galactosidase, MW 116,483.9
50 shots shots high mass low mass
MTMITDSLAV VLQRRDWENP GVTQLNRLAA HPPFASWRNS EEARTDRPSQ QLRSLNGEWR FAWFPAPEAV PESWLECDLP
EADTVVVPSN WQMHGYDAPI YTNVTYPITV NPPFVPTENP TGCYSLTFNV DESWLQEGQT RIIFDGVNSA FHLWCNGRWV
GYGQDSRLPS EFDLSAFLRA GENRLAVMVL RWSDGSYLED QDMWRMSGIF RDVSLLHKPT TQISDFHVAT RFNDDFSRAV
LEAEVQMCGE LRDYLRVTVS LWQGETQVAS GTAPFGGEII DERGGYADRV TLRLNVENPK LWSAEIPNLY RAVVELHTAD
GTLIEAEACD VGFREVRIEN GLLLLNGKPL LIRGVNRHEH HPLHGQVMDE QTMVQDILLM KQNNFNAVRC SHYPNHPLWY
TLCDRYGLYV VDEANIETHG MVPMNRLTDD PRWLPAMSER VTRMVQRDRN HPSVIIWSLG NESGHGANHD ALYRWIKSVD
PSRPVQYEGG GADTTATDII CPMYARVDED QPFAVPKWS IKKWLSLPGE TRPLILCEYA HAMGNSLGGF AKYWQAFRQY
PRLWGGFVWD WVDQSLIKYD ENGNPWSAYG GDFGDTPNDR QFCMNGLVFA DRTPHPALTE AKHQQQFFQF RLSGQTIEVT
SEYLFRHSDN ELLHWMVALD GPKPLASGEVP LDVAPQGKQL IELPELPQPE SAGQLWLTVR VVQPNATAWS EAGHISAWQQ
WRLAENLSVT LPAASHAIPH LTTSEMDFCI ELGNKRWQFN RQSGFLSQMW IGDKKQLLTP LRDQFTRAPL DNDIGVSEAT
RIDPNAWVER WKAAGHYQAE AALLQCTADT LADAVLITTA HAWQHQGKTL FISRKTYRID GSGQMAITVD VEVASDTPPHP
ARIGLNCQLA QVAERVNWLG LGPQENYPDR LTAACFDRWD LPLSDMYTPY VFPSENGLRC GTRELNYGPH QWRGDFQFNI
SRYSQQQLME TSHRHLLHAE EGTWLNIDGF HMGIGGDDSW SPSVSAEFQL SAGRYHYQLV WCQK

17. Missing Peptides MH+ WK
IENGLLLLNGKPLLIR
CSHYPNHPLWYTLCDR
NHPSVIIWSLGNESGHGANHDALYR
DRNHPSVIIWSLGNESGHGANHDALYR

18. 1776

19. IENGLLLLNGKPLLIR Sequence expected
IENGLLLLDGKPLLIR Major component, labeled
IEDGLLLLDGKPLLIR Minor component

20. CSHYPNHPLWYTLCDR
b15-2 S

21. DRNHPSVIIWSLGNDSGHGANHDALYR E MH + = 3001.44
b23
b14
y24
b21

22. Summary of Results 99.8 % of Sequence Covered (1022 of 1024)
AA at position 462 is D not E
Intact disulfide bond at
Deamidation of NG at positions 340 and 346
Discussed by N. E.Robinson PNAS 2002, 99,5283
Dynamic range>1000 and resolving power>10,000 required to obtain good coverage
Results required < 5 min. acquisition time on 125 fmoles loaded (51,000 laser hz)

23. What will the future bring
Laser rates to 10 khz will be routine within two years
A major challenge is developing automated data acquisition, processing, and interpretation systems that can operate continuously at rates in the range of finished spectra/sec.
Applications to protein and peptide samples distributed on surfaces become practical
10,000 discrete spots/sample plate
Multi-channel LC interfaced to single MALDI-TOF
Molecular scanner for 1- and 2-D gels
Direct tissue imaging