Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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.
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.
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.
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.
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.
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.
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.
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.
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.
Acknowledgments
The authors acknowledge the support of the Biotechnology and Biological Sciences Research Council, UK.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2005 Humana Press Inc.
About this protocol
Cite this protocol
Webster, J., Oxley, D. (2005). Peptide Mass Fingerprinting. In: Zanders, E.D. (eds) Chemical Genomics. Methods in Molecular Biology™, vol 310. Humana Press. https://doi.org/10.1007/978-1-59259-948-6_16
Download citation
DOI: https://doi.org/10.1007/978-1-59259-948-6_16
Publisher Name: Humana Press
Print ISBN: 978-1-58829-399-2
Online ISBN: 978-1-59259-948-6
eBook Packages: Springer Protocols