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LC/MS and LC/MS/MS determination of protein tryptic digests

  • Eric C. Huang
  • Jack D. Henion
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Abstract

Tryptic digests were analyzed by means of online microbore liquid chromatography combined with mass spectrometry (LC/MS) for some common proteins. Following conventional enzymatic digestion with trypsin, the freeze-dried residues were dissolved in high-performance liquid chromatography (HPLC) eluent and subjected to gradient reversed-phase microbore HPLC separation with mass spectrometric detection. The latter was done in the full-scan single or tandem (MS/MS) mass spectrometry mode. The formation of gas-phase ions from dissolved analytes was accomplished at atmospheric pressure by pneumatically assisted electrospray (ion spray) ionization. This produced field-assisted ion evaporation of dissolved ions, which could then be mass-analyzed for molecular mass or structure. In the full-scan LC/MS mode, the masses for the peptide fragments in the tryptic digests can be determined as either their singly or multiply charged ions. When the molecular weights of the peptides lie outside the mass range of the mass spectrometer, the multiply charged feature of these experimental conditions still provides reliable molecular weight determinations. In addition, collision-activated dissociation (CAD) on selected peptide precursor ions provides online LC/MS/MS sequence information for the tryptic fragments. Results are shown for the tryptic digests of horse heart cytochrome c, bovine β-lactoglobulin A, and bovine β-lactoglobulin B.

Keywords

Tryptic Fragment Horse Heart Cytochrome Assisted Electrospray Protein Tryptic Digest Microbore HPLC 
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References

  1. 1.
    Arpino, P. J.; Guiochon, G. Anal. Chem. 1979, 51, 682A-701A.CrossRefGoogle Scholar
  2. 2.
    Covey, T. R.; Lee, E. D.; Bruins, A. P.; Henion, J. D. Anal. Chem. 1986, 58, 1451A-1461A.CrossRefGoogle Scholar
  3. 3.
    Garteiz, D. A.; Vestal, M. L. LC Mag. 1985, 3, 334–346.Google Scholar
  4. 4.
    Stachowiak, K.; Wilder, C.; Vestal, M. L.; Dyckes, D. F. J. Am. Chem. Soc. 1988, 110, 1758–1765.CrossRefGoogle Scholar
  5. 5.
    Winkler, P. C.; Perkins, D. D.; Williams, W. K.; Browner, R. F. Anal. Chem. 1988, 60, 489–493.CrossRefGoogle Scholar
  6. 6.
    Ashcroft, A. E. OMS Lett. 1987, 22, 754–757.Google Scholar
  7. 7.
    Caprioli, R. M. Biochemistry 1988, 27, 513–521.CrossRefGoogle Scholar
  8. 8.
    Mosely, A. M.; Deterding, L. J.; Tomer, K. B.; Bragg, N.; Jorgenson, J. W. Capillary zone electrophoresis and capillary liquid chromatography coupled with sector mass spectrometry using coaxial continuous flow FAB interfaces, Proceedings of the 37th ASMS Conference on Mass Spectrometry and Allied Topics; Miami Beach, FL, May 21–26; 1989, p 114.Google Scholar
  9. 9.
    Bourell, J. H.; Henzel, W. J.; Stults, J. T. Analysis of enzymatically digested proteins by on-line capillary HPLC/FABMS, presented at the 37th ASMS Conference on Mass Spectrometry and Allied Topics; Miami Beach, FL, May 21–26, 1989, p 1015.Google Scholar
  10. 10.
    Glajch, J. L. Anal. Chem. 1986, 58, 385–394.CrossRefGoogle Scholar
  11. 11.
    Dong, M. W.; Gant, J. R.; Larsen, B. R. Biochromatography 1989, 4, 19–33.Google Scholar
  12. 12.
    Garnick, R. L.; Solli, N. J.; Papa, P. A. Anal. Chem. 1988, 60, 2546–2557.CrossRefGoogle Scholar
  13. 13.
    Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2301–2303.CrossRefGoogle Scholar
  14. 14.
    Whitehouse, C. M.; Dreyer, R. N.; Yamashita, M.; Fenn, J. B. Anal. Chem. 1985, 57, 675–679.CrossRefGoogle Scholar
  15. 15.
    Mann, M.; Meng, C. K.; Fenn, J. B. Anal. Chem. 1989, 61, 1702–1708.CrossRefGoogle Scholar
  16. 16.
    Covey, T. R.; Bonner, R. F.; Shushan, B. I.; Henion, J. D. Rapid Commun. Mass Spectrom. 1988, 2, 249–255.CrossRefGoogle Scholar
  17. 17.
    Henion, J. D.; Covey, T. R.; Muck; W.; Huang, E. C. LC/MS of proteins and large peptides. Presented at the 37th ASMS Conference on Mass Spectrometry and Allied Topics; Miami Beach, FL, May 21–26, 1989, p 1014.Google Scholar
  18. 18.
    Barinaga, C. J.; Edmonds, C. G.; Udseth, H. R.; Smith, R. D. Rapid Commun. Mass Spectrom. 1989, 3, 160–164.CrossRefGoogle Scholar
  19. 19.
    Smith, R. D.; Loo, J. A.; Barinaga, C. J.; Udseth, H. Presented at the 5th (Montreux) Symposium on LC/MS, Freiburg, FRG, November 1988.Google Scholar
  20. 20.
    Schroeder, W. A. In Handbook of HPLC for the Separation of Amino Acids, Peptides, and Proteins; Hancock, W. S., Ed.; CRC: Boca Raton, FL, Vol. 2, Protein Identification by Peptide Mapping, 1984, pp 287–300.Google Scholar
  21. 21.
    Bruins, A. P.; Covey, T. R.; Henion, J. D. Anal. Chem. 1987, 59, 2642–2646.CrossRefGoogle Scholar
  22. 22.
    Hattangadi, S. LC-GC 1989, 7, 108–114.Google Scholar
  23. 23.
    Lee, E. D.; Covey, T. R.; Henion, J. D. J. Microcol. Sep. 1989, 1, 14–18.CrossRefGoogle Scholar
  24. 24.
    Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984, 11, 601–602.CrossRefGoogle Scholar
  25. 25.
    Hunt, D. F.; Zhu, N.; Shabanowitz, J.; Rapid Commun. Mass Spectrom. 1989, 3, 122–124.CrossRefGoogle Scholar
  26. 26.
    Westmore, J. B.; Ens, W.; Standing, K. G. Biomed. Mass Spectrom. 1982, 9, 119–124.CrossRefGoogle Scholar
  27. 27.
    Barber, M.; Bordoli, R. S.; Sedgwick, D.; Tyler, A. N.; Whalley, E. T. Biomed. Mass Spectrom. 1981, 8, 337–342.CrossRefGoogle Scholar
  28. 28.
    Williams, D. H.; Bradley, C. V.; Santikarn, S.; Bojesen, G. J. Biochem. 1982, 201, 105–117.Google Scholar
  29. 29.
    Konig, W. A.; Aydin, M.; Schulze, U.; Rapp, U.; Hohn, M.; Pesch, R.; Kalikhevitch, V. N. Int, J. Mass Spectrom. Ion Phys. 1983, 46, 403–408.CrossRefGoogle Scholar
  30. 30.
    Gaskell, S. J.; Haroldsen, P. E.; Reilly, M. H. Biomed. Mass Spectrom. 1988, 16, 31–33.CrossRefGoogle Scholar
  31. 31.
    Heerma, W.; Kulik, W. Biomed. Mass Spectrom. 1988, 16, 155–159.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 1990

Authors and Affiliations

  • Eric C. Huang
    • 1
  • Jack D. Henion
    • 1
  1. 1.Drug Testing and ToxicologyCornell UniversityIthacaUSA

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