Large‐scale analysis of the human ubiquitin‐related proteome

Abstract

Protein ubiquitylation contributes to the regulation of many cellular processes including protein degradation, receptor internalization, and repair of DNA damage. We now present a comprehensive characterization of ubiquitin‐conjugated and ubiquitin‐associated proteins in human cells. The proteins were purified by immunoaffinity chromatography under denaturing or native conditions. They were then digested with trypsin, and the resulting peptides were analyzed by 2‐D LC and MS/MS. A total of 670 distinct proteins were identified; 345 proteins (51%) were classified as Urp‐D (ubiquitin‐related proteome under the denaturing condition) and comprised ubiquitin‐conjugated molecules, whereas 325 proteins (49%) were classified as Urp‐N (ubiquitin‐related proteome only under the native condition) and included molecules that associated with ubiquitylated proteins. The proportions of proteins in various functional categories differed substantially between Urp‐D and Urp‐N. Many ribosomal subunits were detected in the Urp‐D group of proteins and several of these subunits were directly shown to be ubiquitylated by mass spectrometric analysis, suggesting that ubiquitylation might play an important role in the regulation and/or quality control of ribosomal proteins. Our results demonstrate the potential of proteomics analysis of protein ubiquitylation to provide important insight into the regulation of protein stability and other ubiquitin‐related cellular functions.