期刊
MOLECULAR & CELLULAR PROTEOMICS
卷 10, 期 2, 页码 -出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/mcp.M110.004796
关键词
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资金
- Canadian Center of Excellence in Commercialization and Research
- Canadian Foundation for Innovation
- Fonds de la Recherche en Sante du Quebec
- National Science and Engineering Research Council
- Canadian Institutes for Health Research
- Canada Research Chair program
- Ligue Nationale Francaise contre le Cancer
- Fonds Quebecois de la Recherche sur la Nature et les Technologies
The small ubiquitin-related modifier (SUMO) is a small group of proteins that are reversibly attached to protein substrates to modify their functions. The large scale identification of protein SUMOylation and their modification sites in mammalian cells represents a significant challenge because of the relatively small number of in vivo substrates and the dynamic nature of this modification. We report here a novel proteomics approach to selectively enrich and identify SUMO conjugates from human cells. We stably expressed different SUMO paralogs in HEK293 cells, each containing a His 6 tag and a strategically located tryptic cleavage site at the C terminus to facilitate the recovery and identification of SUMOylated peptides by affinity enrichment and mass spectrometry. Tryptic peptides with short SUMO remnants offer significant advantages in large scale SUMOylome experiments including the generation of paralog-specific fragment ions following CID and ETD activation, and the identification of modified peptides using conventional database search engines such as Mascot. We identified 205 unique protein substrates together with 17 precise SUMOylation sites present in 12 SUMO protein conjugates including three new sites (Lys-380, Lys-400, and Lys-497) on the protein promyelocytic leukemia. Label-free quantitative proteomics analyses on purified nuclear extracts from untreated and arsenic trioxide-treated cells revealed that all identified SUMOylated sites of promyelocytic leukemia were differentially SUMOylated upon stimulation. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.004796, 1-15, 2011.
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