Advertisement

Detection of Disulfide-Linked Peptides by Mass Spectrometry

  • Alastair Aitken
  • Michèle Learmonth
Protocol
  • 55 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Mass spectrometry is playing a rapidly increasing role in protein chemistry and sequencing (see Chapter 91) and is particularly useful in determining sites of co- and posttranslational modification (1,2), and application in locating disulfide bonds is no exception. This technique can of course readily analyze peptide mixtures. Therefore, it is not always necessary to isolate the constituent peptides. A combination of microsequencing and mass spectrometry techniques is now commonly employed for complete covalent structure determination. On-line electrospray mass spectrometry (ESMS) coupled to capillary electrophoresis or high-performance liquid chromatography (HPLC) has proven particularly valuable in the identification of modified peptides (3). Recent developments in ESMS sources permit on-line microbore HPLC using matrices, such as 10-µm Poros resins slurry-packed into columns <0.25 mm in diameter. Polypeptides can be separated on gradients of 5–75% acetonitrile over 2 min in formic or acetic acid (0.1%) (4).

Keywords

Disulfide Bond Cyanogen Bromide Mass Spectrometry Technique Intramolecular Disulfide Bond Performic Acid 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Burlingame, A. L., Boyd, R. K., and Gaskell, S. J. (1994) Mass spectrometry. Anal. Chem. 66, 634R–683R.PubMedCrossRefGoogle Scholar
  2. 2.
    Mann, M. and Wilm, M. (1995) Electrospray mass spectrometry for protein characterisation. Trends Biochem. Sci. 20, 219–224.PubMedCrossRefGoogle Scholar
  3. 3.
    Kay, I. and Mallet, A. I. (1993) Use of an on-line liquid chromatography trapping column for the purification of protein samples prior to electrospray mass spectrometric analysis Rapid Commun. Mass Specrom. 7, 744–746.CrossRefGoogle Scholar
  4. 4.
    Aitken, A., Howell, S., Jones, D., Madrazo, J., and Patel, Y. (1995) 14-3-3 α and δ are the phosphorylated forms of Raf-activating 14-3-3 β and ζ. In vivo stoichiometric phosphorylation in brain at a Ser-Pro-Glu-Lys motif. J. Biol. Chem. 270, 5706–5709.PubMedCrossRefGoogle Scholar
  5. 5.
    Hunt, D. F., Yates, J. R., Shabanowitz, J., Winston, S., and Hauer, C. R. (1986) Protein sequencing by tandem mass spectrometry. Proc. Natl. Acad. Sci. USA 83, 6233–6237.PubMedCrossRefGoogle Scholar
  6. 6.
    Bean, M. F., Annan, R. S., Hemling, M. E., Mentzer, M., Huddleston, M. J., and Carr, S. A. (1994) LC-MS methods for selective detection of posttranslational modifications in proteins, in Techniques in Protein Chemistry VI (Crabb, J. W., ed.), Academic, New York, pp. 107–116.Google Scholar
  7. 7.
    Morris, R. H. and Pucci, P. (1985). A new method for rapid assignment of S—S bridges in proteins. Biochem. Biophys. Res. Commun. 126, 1122–1128.PubMedCrossRefGoogle Scholar
  8. 8.
    Loo, R., Dales, N., and Andrews, P. C. (1994) Surfactant effects on protein structure examined by electrospray ionisation mass spectrometry. Protein Sci. 3, 1975–1983.PubMedCrossRefGoogle Scholar
  9. 9.
    Vorm, O., Chait, B. T., and Roepstorff, P. (1993) Mass spectrometry of protein samples containing detergents. Proceedings of the 41st ASMS Conference on Mass Spectrometry and Allied Topics, pp. 621,622.Google Scholar
  10. 10.
    Sun, Y. and Smith, D. L. (1988) Identification of disulfide-containing peptides by performic acid oxidation and mass spectrometry. Anal. Biochem. 172, 130–138.PubMedCrossRefGoogle Scholar
  11. 11.
    Chait, B. T., Wang, R., Beavis, R. C., and Kent, S. B. H. (1993) Protein ladder sequencing. Science 262, 89–92.PubMedCrossRefGoogle Scholar
  12. 12.
    Bartlet-Jones, M., Jeffery, W. A. Hansen, H. F., and Pappin, D. J. C. (1994) Peptide ladder sequencing by mass spectrometry using a novel, volatile degradation reagent. Rapid Commun. Mass Spectrom. 8, 737–742.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1996

Authors and Affiliations

  • Alastair Aitken
    • 1
  • Michèle Learmonth
    • 1
  1. 1.National Institute for Medical ResearchMill Hill, LondonUK

Personalised recommendations