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Protein abundance and phosphorylation convey important information about pathway activity and molecular pathophysiology in diseases including cancer, providing biological insight, informing drug and diagnostic development, and guiding therapeutic intervention. Analyzed tissues are usually collected without tight regulation or documentation of ischemic time. To evaluate the impact of ischemia, we collected human ovarian tumor and breast cancer xenograft tissue without vascular interruption and performed quantitative proteomics and phosphoproteomics after defined ischemic intervals. Although the global expressed proteome and most of the >25,000 quantified phosphosites were unchanged after 60 min, rapid phosphorylation changes were observed in up to 24% of the phosphoproteome, representing activation of critical cancer pathways related to stress response, transcriptional regulation, and cell death. Both pan-tumor and tissue-specific changes were observed. The demonstrated impact of pre-analytical tissue ischemia on tumor biology mandates caution in interpreting stress-pathway activation in such samples and motivates reexamination of collection protocols for phosphoprotein analysis., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

We report a mass spectrometry-based method for the integrated analysis of protein expression, phosphorylation, ubiquitination and acetylation by serial enrichments of different post-translational modifications (SEPTM) from the same biological sample. This technology enabled quantitative analysis of nearly 8,000 proteins and more than 20,000 phosphorylation, 15,000 ubiquitination and 3,000 acetylation sites per experiment, generating a holistic view of cellular signal transduction pathways as exemplified by analysis of bortezomib-treated human leukemia cells.

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.