Summary
Identification and characterization of proteins are ultimately the goal in proteomic analysis. In order to identify a protein trypsin is commonly used to digest protein into peptides which can be analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS). This chapter describes a tryptic digestion method for digestion of proteins in one-dimensional (1DE) or two-dimensional (2DE) polyacrylamide gels. The method involves cutting target protein bands or spots, removal of protein stain, reduction and alkylation of native protein, digestion and finally extraction of peptides for mass spectrometry analysis. The method is simple and reasonably sensitive that many in-gel proteins that are barely visible with Coomassie blue stain have been successfully identified.
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
Kellner, R. (1994) Chemical and Enzymatic Fragmentation of Proteins. In Microcharacterization of Proteins (Kellner, R., ed.), pp. 11–27. VCH, New York
Henzel, W. J., and Watanabe, C. (2003) Protein identification: the origins of peptide mass fingerprinting. J. Am. Soc. Mass Spectrom. 14, 931–942
Yates, J.R. III, Speiche, S. Griffin, P.R., and Hunkapiller, T. (1993) Peptide mass maps: a highly informatic approach to protein identification. Anal. Biochem. 214, 397–408
Yates, J.R. III, Eng, J.K., McCormack, A.L.,and Schieltz, D. (1995) Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Anal. Chem. 67, 1426–1436
Westermeier, R., and Naven, T. (2002) Proteomics in Practice. Wiley-VCH Weinheim Germany
Wilm, M., Shevchenko A., Houthaeve T., Breit, S., Scheweigerer, L., Fotsis, T., and Mann, M. (1996) Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379, 466–469
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
Kellner, R., Lottspeigh, F., and Meyer, H.E. (ed.) (1994) Microcharacterizaion of Proteins. VCH, NY
Herbert, B., Galvani, M., Hamdan, M., Olivieri, E., MacCarthy, J., Pedersen, S., and Righetti, P. G. (2001) Reduction and alkylation of proteins in preparation of two-dimensional map analysis: whywhenand how? Electrophoresis 22, 2046–2205
Herbert, B., Molloy, M.P., Gooley, A.A., Walsh, B.J., Bryson, W.G., and Williams, K.L., (1998) Improved protein solubility in two-dimensional-gel electrophoresis using tributyl phosphine as a reducing agent. Electrophoresis 19, 845–851
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Huynh, ML., Russell, P., Walsh, B. (2009). Tryptic Digestion of In-Gel Proteins for Mass Spectrometry Analysis. In: Tyther, R., Sheehan, D. (eds) Two-Dimensional Electrophoresis Protocols. Methods in Molecular Biology, vol 519. Humana Press. https://doi.org/10.1007/978-1-59745-281-6_34
Download citation
DOI: https://doi.org/10.1007/978-1-59745-281-6_34
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-58829-937-6
Online ISBN: 978-1-59745-281-6
eBook Packages: Springer Protocols