Multiple functional categories of proteins identified in an in vitro cellular ubiquitin affinity extract using shotgun peptide sequencing.

To construct a high information content assay for examination of the function of the cellular ubiquitin system, we added his-tagged ubiquitin, ATP, and an ATP-regenerating system to endogenous human cellular ubiquitin system enzymes, and labeled cellular proteins with hexa-histidine tagged ubiquitin in vitro. Labeling depended on ATP, the ATP recycling system, the proteasome inhibitor MG132, and the ubiquitin protease inhibitor ubiquitin aldehyde, and was inhibited by iodoacetamide. Quadruplicate affinity extracted proteins were digested with trypsin, and the peptides were analyzed by 2D capillary LC−MS/MS, SEQUEST, MEDUSA, and support vector machine calculations. Identified proteins included 22 proteasome subunits or associated proteins, 18 E1, E2, or E3 ubiquitin system enzymes or related proteins, 4 ubiquitin domain proteins and 36 proteins in functional clusters associated with redox processes, endocytosis/vesicle trafficking, the cytoskeleton, DNA damage/repair, calcium binding, and mRNA splicing. Th...