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Detecting endogenous SUMO targets in mammalian cells and tissues
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Detecting endogenous SUMO targets in mammalian cells and tissues
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Detecting endogenous SUMO targets in mammalian cells and tissues
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Journal Article
Detecting endogenous SUMO targets in mammalian cells and tissues
2013
Overview
SUMOylation is a dynamic protein post-translational modification that regulates many eukaryotic proteins. Now a methodology using commercially available monoclonal antibodies coupled to MS analysis leads to the enrichment and identification of endogenous targets for SUMO1 and for SUMO2/3 in HeLa cells and mouse liver. This protocol can be adapted for other tissues and organs.
SUMOylation is an essential modification that regulates hundreds of proteins in eukaryotic cells. Owing to its dynamic nature and low steady-state levels, endogenous SUMOylation is challenging to detect. Here, we present a method that allows efficient enrichment and identification of endogenous targets of SUMO1 and the nearly identical SUMO2 and 3 (SUMO 2/3) from vertebrate cells and complex organ tissue. Using monoclonal antibodies for which we mapped the epitope, we enriched SUMOylated proteins by immunoprecipitation and peptide elution. We used this approach in combination with MS to identify SUMOylated proteins, which resulted in the first direct comparison of the endogenous SUMO1- and SUMO2/3-modified proteome in mammalian cells, to our knowledge. This protocol provides an affordable and feasible tool to investigate endogenous SUMOylation in primary cells, tissues and organs, and it will facilitate understanding of SUMO's role in physiology and disease.
Publisher
Nature Publishing Group US,Nature Publishing Group
Subject
/ Analysis
/ Animals
/ Antibodies, Monoclonal - immunology
/ Electrophoresis, Polyacrylamide Gel
/ Humans
/ Mammals
/ Proteins
/ Sequence Homology, Amino Acid
/ Small Ubiquitin-Related Modifier Proteins - chemistry
/ Small Ubiquitin-Related Modifier Proteins - immunology
/ Small Ubiquitin-Related Modifier Proteins - metabolism
/ Tissues
