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.