Large-scale identification of ubiquitination sites by mass spectrometry

Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
Nature Protocol (Impact Factor: 9.67). 10/2013; 8(10):1950-60. DOI: 10.1038/nprot.2013.120
Source: PubMed


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.

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    • "To overcome this limitation, we decided to enrich the ubiquitinated peptides prior to mass spectrometry analysis using a two-step immunoprecipitation protocol. A mixture of light-labeled Flag-VAPB and heavy-labeled Flag-FAF1 immunoprecipitates was trypsin-digested and the peptides carrying a di-glycine signature were isolated using antibodies specific to lysine-ϵ-GlyGly [37]. Di-glycine peptides mark the site of ubiquitination in the protein of origin. "
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