Peptide Mass Fingerprinting

Protein Identification Using MALDI-TOF Mass Spectrometry
  • Judith Webster
  • David Oxley
Part of the Methods in Molecular Biology™ book series (MIMB, volume 310)


Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) is now routinely used in many laboratories for the rapid and sensitive identification of proteins by peptide mass fingerprinting (PMF). We describe a simple protocol that can be performed in a standard biochemistry laboratory, whereby proteins separated by one- or two-dimensional gel electrophoresis can be identified at femtomole levels. The procedure involves excision of the spot or band from the gel, washing and de-staining, reduction and alkylation, in-gel trypsin digestion, MALDI-TOF MS of the tryptic peptides, and database searching of the PMF data. Up to 96 protein samples can easily be manually processed at one time by this method.

Key Words

Proteomics MALDI-TOF mass spectrometry SDS-PAGE 2D-gel in-gel digestion peptide mass fingerprint protein identification database searching 



The authors acknowledge the support of the Biotechnology and Biological Sciences Research Council, UK.


  1. 1.
    Pappin, D. J. C., Hojrup, P., and Bleasby, A. J. (1993) Rapid identification of proteins by peptide-mass fingerprinting. Curr. Biol. 3(6), 327–332.PubMedCrossRefGoogle Scholar
  2. 2.
    Neuhoff, V., Stamm, R., and Eibl, H. (1985) Clear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels: a systematic analysis. Electrophoresis 6, 427–448.CrossRefGoogle Scholar
  3. 3.
    Herbert, B., Galvani, M., Hamdan, M., et al. (2001) Reduction and alkylation of proteins in preparation of two-dimensional map analysis: why, when, and how? Electrophoresis 22, 2046–2057.PubMedCrossRefGoogle Scholar
  4. 4.
    Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858.PubMedCrossRefGoogle Scholar
  5. 5.
    Gharahdaghi, F., Weinberg, C. R., Meagher, D. A., and Imai, B. S. (1999) Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. Electrophoresis 20, 601–605.PubMedCrossRefGoogle Scholar
  6. 6.
    Sechi, S. and Chait, B. T. (1998) Modification of cysteine residues by alkylation. A tool in peptide mapping and protein identification. Anal. Chem. 70, 5150–5158.PubMedCrossRefGoogle Scholar
  7. 7.
    Harris, W. A., Janecki, D. J., and Reilly, J. P. (2002) Use of matrix clusters and trypsin autolysis fragments as mass calibrants in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 16, 1714–1722.PubMedCrossRefGoogle Scholar
  8. 8.
    Karty, J. A., Ireland, M. M. E., Brun, Y. V., and Reilly, J. P. (2002) Artifacts and unassigned masses encountered in peptide mass mapping. J. Chrom. B 782, 363–383.CrossRefGoogle Scholar
  9. 9.
    Schmidt, F., Schmid, M., Jungblut, P. R., Mattow, J., Facius, A., and Pleissner, K. P. (2003) Iterative data analysis is the key for exhaustive analysis of peptide mass fingerprints from proteins separated by two-dimensional electrophoresis. J. Am. Soc. Mass Spectrom. 14, 943–956.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Judith Webster
    • 1
  • David Oxley
    • 1
  1. 1.Proteomics Research GroupBabraham InstituteCambridgeUK

Personalised recommendations