Abstract
MALDI-TOF mass spectrometers are now commonplace and their relative ease of use means that most non-specialist labs can readily access the technology for the rapid and sensitive analysis of biomolecules. One of the main uses of MALDI-TOF-MS is in the identification of proteins, by peptide mass fingerprinting (PMF). Here we describe a simple protocol that can be performed in a standard biochemistry laboratory, whereby proteins separated by 1D or 2D gel electrophoresis can be identified at femtomole levels. The procedure involves excision of the spot or band from the gel, washing and destaining, 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.
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References
Pappin DJC, Hojrup P, Bleasby, AJ (1993) Rapid identification of proteins by peptide-mass fingerprinting. Current Biology 3:327–332
Neuhoff V, Stamm R, 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, 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, et al (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68:850–858
Gharahdaghi F, et al (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, Chait BT (1998) Modification of cysteine residues by alkylation. A tool in peptide mapping and protein identification. Anal Chem 70:5150–5158
Harris WA, Janecki DJ, Reilly JP (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 JA, et al (2002) Artifacts and unassigned masses encountered in peptide mass mapping. J Chrom B 782:363–383
Schmidt F, et al (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.
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Webster, J., Oxley, D. (2012). Protein Identification by MALDI-TOF Mass Spectrometry. In: Zanders, E. (eds) Chemical Genomics and Proteomics. Methods in Molecular Biology, vol 800. Humana Press. https://doi.org/10.1007/978-1-61779-349-3_15
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DOI: https://doi.org/10.1007/978-1-61779-349-3_15
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Publisher Name: Humana Press
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Online ISBN: 978-1-61779-349-3
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