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

Detection of endogenous ubiquitination sites by mass spectrometry has dramatically improved with the commercialization of anti-di-glycine remnant (K-epsilon-GG) antibodies. Here, we describe a number of improvements to the K-epsilon-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 similar to 20,000 distinct endogenous ubiquitination sites in a single SILAC experiment using moderate amounts of protein input. Molecular & Cellular Proteomics 12: 10.1074/mcp.O112.027094, 825-831, 2013.