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

Tarikere Gururaja, Weiqun Li, William Stafford Noble, Donald G. Payan and D. C. Anderson

Journal of Proteome Research. 2:394-404, 2003.


Using endogenous human cellular ubiquitin system enzymes and added his-tagged ubiquitin, ATP, and an ATP-regenerating system, we labelled cellular proteins with hexahistidine tagged ubiquitin in vitro. Labeling was dependent on ATP and the ATP recycling system, on the proteasome inhibitor MG132 and the ubiquitin protease inhibitor ubiquitin aldehyde, and was inhibited by iodoacetamide. Labeled proteins were affinity extracted in quadruplicate and tryptic peptides identifed by 2D capillary LC/MS/MS comb9ined with SEQUEST and MEDUSA analyses. Support vector machine analyais of the mass spectrometry data allowed prediction of correct matches between mass spectrometry data and peptide sequences. Overall, 144 proteins were identified by peptides predicted to be correctly sequenced, and 113 were identified by at least three peptides or one or two peptides with at least an 80% chance of being correct. Identified proteins included 22 proteasome subunits or associated proteins, 18 E1, E2 or E3 ubiquitin system enzymes or related proteins, and four ubiquitin domain proteins. Seventeen directly ubiquitinated proteins or proteins associated with the ubiquitin system were identified. Functional clusters of other proteins included redox enzymes, proteins associated with endocytosis, cytoskeletal proteins, DNA damage or repair related proteins, calcium binding proteins, and splicing factor and related proteins, suggesting that in vitro ubiquitination is not random, and that these functions may be regulated by the ubiquitin system. This map of cellular ubiquitinated proteins and their interacting proteins will be useful for further studies of ubiquitin system function.