Large-scale identification of ubiquitination sites by mass spectrometry.

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.