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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

Item Type:Article
Title: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
Creators Name:Udeshi, N.D. and Svinkina, T. and Mertins, P. and Kuhn, E. and Mani, D.R. and Qiao, J.W. and Carr, S.A.
Abstract: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 ~20,000 distinct endogenous ubiquitination sites in a single SILAC experiment using moderate amounts of protein input.
Keywords:Amino Acids, Antibodies, Binding Sites, Cross-Linking Reagents, Cysteine Proteinase Inhibitors, Glycylglycine, Isotope Labeling, Jurkat Cells, Leupeptins, Liquid Chromatography, Proteasome Endopeptidase Complex, Proteome, Proteomics, Reproducibility of Results, Tandem Mass Spectrometry, Ubiquitinated Proteins, Ubiquitination
Source:Molecular & Cellular Proteomics
ISSN:1535-9476
Publisher:American Society for Biochemistry and Molecular Biology (U.S.A.)
Volume:12
Number:3
Page Range:825-831
Date:1 March 2013
Official Publication:https://doi.org/10.1074/mcp.O112.027094
PubMed:View item in PubMed

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