Quantitative Analysis of global Ubiquitination in HeLa Cells by Mass Spectrometry

摘要: Ubiquitination regulates a host of cellular processes by labeling proteins for degradation, but also functioning as regulatory, nonproteolytic posttranslational modification. Proteome-wide strategies to monitor changes in ubiquitination profiles are important obtain insight into the various functions ubiquitination. Here we describe generation stable cell lines expressing tandem hexahistidine-biotin tag (HB-tag) fused ubiquitin two-step purification ubiquitinated proteome under fully denaturing conditions. Using this approach identified 669 from HeLa cells, including 44 precise attachment sites on substrates and all seven possible chain-linkage types. To probe dynamics response perturbation ubiquitin/proteasome pathway, combined profiling with quantitative mass spectrometry using isotope amino acids culture (SILAC) strategy. We compared untreated cells treated proteasome inhibitor MG132 identify that targeted degradation. A number were identified. In addition, allowed us compare targeting different chain topologies vivo. The tools described here can be applied detect growth conditions stress will contribute our understanding system.