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Molecular & cellular proteomics : MCP

Refined preparation and use of anti-diglycine remnant (K-ε-GG) antibody enables routine quantification of 10,000s of ubiquitination sites in single proteomics experiments.


PMID 23266961

Abstract

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