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Nat Protoc DOI:10.1038/nprot.2013.120

Large-scale identification of ubiquitination sites by mass spectrometry.

Publication TypeJournal Article
Year of Publication2013
AuthorsUdeshi, ND, Mertins, P, Svinkina, T, Carr, SA
JournalNat Protoc
Volume8
Issue10
Pages1950-60
Date Published2013 Oct
ISSN1750-2799
KeywordsCells, Cultured, Chemical Fractionation, Chromatography, Reverse-Phase, Mass Spectrometry, Ubiquitin, Ubiquitination
Abstract

Ubiquitination is essential for the regulation of cellular protein homeostasis. It also has a central role in numerous signaling events. Recent advances in the production and availability of antibodies that recognize the Lys-ɛ-Gly-Gly (K-ɛ-GG) remnant produced by trypsin digestion of proteins having ubiquitinated lysine side chains have markedly improved the ability to enrich and detect endogenous ubiquitination sites by mass spectrometry (MS). The following protocol describes the steps required to complete a large-scale ubiquitin experiment for the detection of tens of thousands of distinct ubiquitination sites from cell lines or tissue samples. Specifically, we present detailed, step-by-step instructions for sample preparation, off-line fractionation by reversed-phase chromatography at pH 10, immobilization of an antibody specific to K-ɛ-GG to beads by chemical cross-linking, enrichment of ubiquitinated peptides using these antibodies and proteomic analysis of enriched samples by LC-tandem MS (MS/MS). Relative quantification can be achieved by performing stable isotope labeling by amino acids in cell culture (SILAC) labeling of cells. After cell or tissue samples have been prepared for lysis, the described protocol can be completed in ∼5 d.

URLhttp://dx.doi.org/10.1038/nprot.2013.120
DOI10.1038/nprot.2013.120
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/24051958?dopt=Abstract

Alternate JournalNat Protoc
PubMed ID24051958
PubMed Central IDPMC4725055
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 / / PHS HHS / United States
HHSN268201000033C / HL / NHLBI NIH HHS / United States
U24CA160034 / CA / NCI NIH HHS / United States