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.
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.
authors.affiliation: Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, MA 02142, USA. udeshi@broadinstitute.org
authors.forename: Namrata D
authors.initials: ND
authors.lastname: Udeshi
authors.forename: Tanya
authors.initials: T
authors.lastname: Svinkina
authors.forename: Philipp
authors.initials: P
authors.lastname: Mertins
authors.forename: Eric
authors.initials: E
authors.lastname: Kuhn
authors.forename: D R
authors.initials: DR
authors.lastname: Mani
authors.forename: Jana W
authors.initials: JW
authors.lastname: Qiao
authors.forename: Steven A
authors.initials: SA
authors.lastname: Carr
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D001665:Binding Sites; D002853:Chromatography, Liquid / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006033:Glycylglycine / Q000276:immunology; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D019169:Jurkat Cells; D007976:Leupeptins / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods; D057149:Ubiquitinated Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D000596:Amino Acids; D000906:Antibodies; D003432:Cross-Linking Reagents; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D020543:Proteome; D057149:Ubiquitinated Proteins; D006033:Glycylglycine; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde