期刊
NATURE PROTOCOLS
卷 12, 期 11, 页码 2342-2358出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2017.105
关键词
-
资金
- Natural Sciences and Engineering Research Council of Canada (NSERC)
- Genome Canada
- NSERC
- faculty of graduate studies of the Universite de Montreal
Ubiquitin and ubiquitin-like modifiers (UBLs) such as small ubiquitin-like modifier (SUSUMO) can act as antagonists to one another by competing to occupy similar residues in the proteome. In addition, SUSUMO and ubiquitin can be coupled to each other at key lysine residues to form highly branched protein networks. The interplay between these modifications governs important biological processes such as double-strand break repair and meiotic recombination. We recently developed an approach that permits the identification of proteins that are modified by both SUSUMOylation and ubiquitylation. This protocol requires cells that express a mutant 6xHis-SUSUMO3m protein that has had its C terminus modified from QQQTGG to RNRNQTGG, enabling the purification of SUSUMOylated peptides and their identification by tandem mass spectrometry (MS/MS). Cells are lysed under denaturing conditions, and the SUSUMOylated proteins are purified on nickel-nitrilotriacetic acid (Ni-NTANTANTA) resin via the 6xHis on the SUSUMO3m construct. After on-bead digestion using trypsin, ubiquitylated peptides are enriched by immunoprecipitation, and the flow-through from this step is subjected to anti-SUSUMO immunoprecipitation. The SUSUMOylated peptides are fractionated on strong cation exchange (SCSCX) StageTips to enhance the coverage of the SUSUMO proteome. The ubiquitylated and SUSUMOylated peptides are analyzed separately by liquid chromatography (LCLC)-MS/MS and identified with MaxQuant. We demonstrate how this approach can be used to identify temporal changes in SUSUMOylated and ubiquitylated proteins in response to, for instance, heat shock and proteasome inhibition. The procedure requires 3 d when starting from cell pellets and yields >8,000 SUSUMO sites and >3,500 ubiquitin sites from 16 mg of cell extract.
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