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Molecular & cellular proteomics : MCP DOI:10.1074/mcp.O112.027094

Refined Preparation and Use of Anti-K- epsilon -GG Antibody Enables Routine Quantification of 10,000s of Ubiquitination Sites in Single Proteomics Experiments.

Publication TypeJournal Article
Year of Publication2012
AuthorsUdeshi, ND, Svinkina, T, Mertins, P, Kuhn, E, Mani, DR, Qiao, JW, Carr, SA
JournalMolecular & cellular proteomics : MCP
Date Published2012/12/24

Detection of endogenous ubiquitination sites by mass spectrometry has dramatically improved with the commercialization of anti-K-ε-GG remnant antibodies. Here we describe a number of improvements to the K-ε-GG enrichment workflow including optimized antibody and peptide input requirements, antibody crosslinking, and improved offline fractionation prior to enrichment. This refined and practical workflow enables routine identification and quantification of approximately 20,000 distinct endogenous ubiquitination sites in a single SILAC experiment using moderate amounts of protein input.