A Chemical and Enzymatic Approach to Study Site-Specific Sumoylation.

ABSTRACT: A variety of cellular pathways are regulated by protein modifications with ubiquitin-family proteins. SUMO, the Small Ubiquitin-like MOdifier, is covalently attached to lysine on target proteins via a cascade reaction catalyzed by E1, E2, and E3 enzymes. A major barrier to understanding the diverse regulatory roles of SUMO has been a lack of suitable methods to identify protein sumoylation sites. Here we developed a mass-spectrometry (MS) based approach combining chemical and enzymatic modifications to identify sumoylation sites. We applied this method to analyze the auto-sumoylation of the E1 enzyme in vitro and compared it to the GG-remnant method using Smt3-I96R as a substrate. We further examined the effect of smt3-I96R mutation in vivo and performed a proteome-wide analysis of protein

SUBMITTER: Albuquerque CP 

PROVIDER: S-EPMC4669148 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature