Abstract
In-gel digestion of protein spots derived from two-dimensional gels and their subsequent identification by mass spectrometry is involved in a multitude of mass spectrometry-driven proteomic experiments, including fluorescence difference gel electrophoresis (DIGE). This type of proteomic methodology has been involved in the establishment of comparative proteome maps and in the identification of differentially expressed proteins and protein isoforms in health and disease. Most in-gel digestion protocols follow a number of common steps including excision of the protein spots of interest, de-staining, reduction and alkylation (for silver-stained gels), dehydration and overnight digestion with the proteolytic enzyme of choice. While trypsin has been a mainstay of peptide digestion for many years, it does have its shortcomings, particularly related to incomplete peptide digestion, and this has led to a rise in popularity for other proteolytic enzymes either used alone or in combination. This chapter discusses the alternative enzymes available and describes the process of in-gel digestion using the enzyme trypsin.
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Acknowledgements
Research in the author’s laboratory has been supported by project grants from the Irish Higher Education Authority, the Irish Health Research Board and Muscular Dystrophy Ireland, as well as a Hume Scholarship from Maynooth University.
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Murphy, S., Ohlendieck, K. (2018). Protein Digestion for DIGE Analysis. In: Ohlendieck, K. (eds) Difference Gel Electrophoresis. Methods in Molecular Biology, vol 1664. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7268-5_17
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DOI: https://doi.org/10.1007/978-1-4939-7268-5_17
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