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Mol Cell Proteomics DOI:10.1074/mcp.O112.027094

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.

Publication TypeJournal Article
Year of Publication2013
AuthorsUdeshi, ND, Svinkina, T, Mertins, P, Kuhn, E, Mani, DR, Qiao, JW, Carr, SA
JournalMol Cell Proteomics
Volume12
Issue3
Pages825-31
Date Published2013 Mar
ISSN1535-9484
KeywordsAmino Acids, Antibodies, Binding Sites, Chromatography, Liquid, Cross-Linking Reagents, Cysteine Proteinase Inhibitors, Glycylglycine, Humans, Isotope Labeling, Jurkat Cells, Leupeptins, Proteasome Endopeptidase Complex, Proteome, Proteomics, Reproducibility of Results, Tandem Mass Spectrometry, Ubiquitinated Proteins, Ubiquitination
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.

URLhttp://www.mcponline.org/cgi/pmidlookup?view=long&pmid=23266961
DOI10.1074/mcp.O112.027094
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/23266961?dopt=Abstract

Alternate JournalMol. Cell Proteomics
PubMed ID23266961
PubMed Central IDPMC3591673
Grant ListU24 CA160034 / CA / NCI NIH HHS / United States
R01HL096738 / HL / NHLBI NIH HHS / United States
R01 HL096738 / HL / NHLBI NIH HHS / United States
HHSN268201000033C / HL / NHLBI NIH HHS / United States
U24CA160034 / CA / NCI NIH HHS / United States