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Mol Cell Proteomics. 2013 Mar;12(3):825-31. doi: 10.1074/mcp.O112.027094. Epub 2012 Dec 24.

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.

Author information

1
Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, MA 02142, USA. udeshi@broadinstitute.org

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.

PMID:
23266961
PMCID:
PMC3591673
DOI:
10.1074/mcp.O112.027094
[Indexed for MEDLINE]
Free PMC Article

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