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The Future of Time-of-flight Mass Spectrometry

The Future of Time-of-flight Mass Spectrometry

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The Future of Time-of-flight Mass Spectrometry

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  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. JETP37, 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, RCM10, 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-TOFyesterday, today, and tomorrow then now future Laser Rate (hz) 2 200 10,000 Acq. Time/Spect.(sec) 60 2 0.1 Spectra/day 1000 40,000 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 cm2 @100 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 1 11 21 31 41 51 61 71 MTMITDSLAV VLQRRDWENP GVTQLNRLAA HPPFASWRNS EEARTDRPSQ QLRSLNGEWR FAWFPAPEAV PESWLECDLP 81 91 101 111 121 131 141 151 EADTVVVPSN WQMHGYDAPI YTNVTYPITV NPPFVPTENP TGCYSLTFNV DESWLQEGQT RIIFDGVNSA FHLWCNGRWV 161 171 181 191 201 211 221 231 GYGQDSRLPSEFDLSAFLRA GENRLAVMVL RWSDGSYLED QDMWRMSGIF RDVSLLHKPT TQISDFHVATRFNDDFSRAV 241 251 261 271 281 291 301 311 LEAEVQMCGE LRDYLRVTVSLWQGETQVASGTAPFGGEIIDERGGYADRV TLRLNVENPK LWSAEIPNLY RAVVELHTAD 321 331 341 351 361 371 381 391 GTLIEAEACD VGFREVRIEN GLLLLNGKPL LIRGVNRHEH HPLHGQVMDE QTMVQDILLM KQNNFNAVRC SHYPNHPLWY 401 411 421 431 441 451 461 471 TLCDRYGLYV VDEANIETHG MVPMNRLTDD PRWLPAMSER VTRMVQRDRN HPSVIIWSLG NESGHGANHD ALYRWIKSVD 481 491 501 511 521 531 541 551 PSRPVQYEGG GADTTATDII CPMYARVDED QPFAVPKWS IKKWLSLPGE TRPLILCEYA HAMGNSLGGF AKYWQAFRQY 561 571 581 591 601 611 621 631 PRLWGGFVWD WVDQSLIKYD ENGNPWSAYG GDFGDTPNDR QFCMNGLVFA DRTPHPALTE AKHQQQFFQF RLSGQTIEVT 641 651 661 671 681 691 701 711 SEYLFRHSDN ELLHWMVALD GPKPLASGEVP LDVAPQGKQL IELPELPQPE SAGQLWLTVRVVQPNATAWS EAGHISAWQQ 721 731 741 751 761 771 781 791 WRLAENLSVT LPAASHAIPH LTTSEMDFCI ELGNKRWQFN RQSGFLSQMW IGDKKQLLTP LRDQFTRAPL DNDIGVSEAT 801 811 821 831 841 851 861 871 RIDPNAWVER WKAAGHYQAE AALLQCTADT LADAVLITTA HAWQHQGKTL FISRKTYRIDGSGQMAITVDVEVASDTPPHP 881 891 901 911 921 931 941 951 ARIGLNCQLA QVAERVNWLG LGPQENYPDR LTAACFDRWD LPLSDMYTPY VFPSENGLRC GTRELNYGPH QWRGDFQFNI 961 971 981 991 1001 1011 1021 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) 975-1014 563-600

  13. 10,000 laser shots Focused at low mass (50 seconds) 290-293 VTLR ? 335-337 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 1000-3000 100 18 14 78 19 1.65 9.0 30 29 23 79 36 1.56 8.3 10 49 33 67 45 1.07 8.9 3 196 52 27 53 0.60 15.0 3000-10000 10 26 9 35 31 -2.18 10.8 400-1000 30 7 5 71 3 -7.58 13.9 10 18 11 61 5 -6.02 9.8 3 156 24 15 12 -5.01 15.4 Total 3 378 85 23 96

  15. M/z 1000-3000, 1000 laser shots Peaks detected down to S/N =3 52/196 matched Number of Peptides Matched -20 -18 -16 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 18 Error (ppm)

  16. E Coli beta-galactosidase, MW 116,483.9 50 shots1000 shotshigh masslow mass 1 11 21 31 41 51 61 71 MTMITDSLAV VLQRRDWENP GVTQLNRLAAHPPFASWRNS EEARTDRPSQ QLRSLNGEWR FAWFPAPEAVPESWLECDLP 81 91 101 111 121 131 141 151 EADTVVVPSN WQMHGYDAPI YTNVTYPITV NPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVNSA FHLWCNGRWV 161 171 181 191 201 211 221 231 GYGQDSRLPSEFDLSAFLRAGENRLAVMVLRWSDGSYLED QDMWRMSGIFRDVSLLHKPT TQISDFHVATRFNDDFSRAV 241 251 261 271 281 291 301 311 LEAEVQMCGELRDYLRVTVSLWQGETQVASGTAPFGGEIIDERGGYADRV TLRLNVENPK LWSAEIPNLY RAVVELHTAD 321 331 341 351 361 371 381 391 GTLIEAEACDVGFREVRIEN GLLLLNGKPL LIRGVNRHEH HPLHGQVMDEQTMVQDILLMKQNNFNAVRC SHYPNHPLWY 401 411 421 431 441 451 461 471 TLCDRYGLYV VDEANIETHG MVPMNRLTDDPRWLPAMSER VTRMVQRDRN HPSVIIWSLG NESGHGANHD ALYRWIKSVD 481 491 501 511 521 531 541 551 PSRPVQYEGG GADTTATDII CPMYARVDEDQPFAVPKWSIKKWLSLPGETRPLILCEYA HAMGNSLGGFAKYWQAFRQY 561 571 581 591 601 611 621 631 PRLWGGFVWD WVDQSLIKYD ENGNPWSAYGGDFGDTPNDR QFCMNGLVFA DRTPHPALTEAKHQQQFFQF RLSGQTIEVT 641 651 661 671 681 691 701 711 SEYLFRHSDNELLHWMVALDGPKPLASGEVP LDVAPQGKQL IELPELPQPE SAGQLWLTVRVVQPNATAWS EAGHISAWQQ 721 731 741 751 761 771 781 791 WRLAENLSVT LPAASHAIPH LTTSEMDFCI ELGNKRWQFN RQSGFLSQMWIGDKKQLLTP LRDQFTRAPL DNDIGVSEAT 801 811 821 831 841 851 861 871 RIDPNAWVER WKAAGHYQAEAALLQCTADTLADAVLITTAHAWQHQGKTLFISRKTYRIDGSGQMAITVDVEVASDTPPHP 881 891 901 911 921 931 941 951 ARIGLNCQLA QVAERVNWLG LGPQENYPDR LTAACFDRWDLPLSDMYTPY VFPSENGLRCGTRELNYGPH QWRGDFQFNI 961 971 981 991 1001 1011 1021 SRYSQQQLMETSHRHLLHAE EGTWLNIDGFHMGIGGDDSWSPSVSAEFQLSAGRYHYQLVWCQK

  17. Missing Peptides MH+ 811-812 WK 333. 338-353 IENGLLLLNGKPLLIR 1776.111 390-405 CSHYPNHPLWYTLCDR 2004.885 450-474 NHPSVIIWSLGNESGHGANHDALYR 2744.329 448-474 DRNHPSVIIWSLGNESGHGANHDALYR 3015.457

  18. 1776 257-283 358-381

  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 390-403 • 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 shots @200 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 10-100 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