Abstract
SUMOylation is a reversible post-translational modification involved in various critical biological processes. To date, there is limited approach for endogenous wild-type SUMO-modified peptides enrichment and SUMOylation sites identification. In this study, we generated a high-affinity SUMO1 antibody to facilitate the enrichment of endogenous SUMO1-modified peptides from Trypsin/Lys-C protease digestion. Following secondary Glu-C protease digestion, we identified 53 high-confidence SUMO1-modified sites from mouse testis by using high-resolution mass spectrometry. Bioinformatics analyses showed that SUMO1-modified proteins were enriched in transcription regulation and DNA repair. Nab1 was validated to be an authentic SUMOylated protein and Lys479 was identified to be the major SUMOylation site. The SUMOylation of Nab1 enhanced its interaction with HDAC2 and maintained its inhibitory effect on EGR1 transcriptional activity. Therefore, we provided a novel approach to investigating endogenous SUMOylation sites in tissue samples.
Highlights
From the ‡State Key Laboratory of Genetic Engineering and Collaborative Innovation Center for Genetics and Development, Institutes of Biomedical Sciences, School of Life Sciences, Fudan University, Shanghai 200032, China; §State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine; National Center for Protein Sciences (The PHOENIX center, Beijing), Beijing 102206, China; ¶Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
We validated the SUMOylation site of Nab1 identified in this study by immunoprecipitation (IP) assay, and revealed that K480R mutant NAB1 impaired its interaction with HDAC2 and attenuated the inhibitory effect of wild-type NAB1 on EGR1 transcriptional activity
As the remnant SUMO1 chain is too long to be identified by mass spectrometry (MS), we executed the second enzyme digestion with Glu-C to obtain a much shorter remnant SUMO1 side chain (QTGG), facilitating MS identification for endogenous SUMO1modified sites
Summary
This antibody was used for the identification of endogenous SUMO1 modification sites of mouse testis tissue. Combining all the results from 36 raw files, 132 putative SUMO1-modified sites were identified in mouse testis by MaxQuant, of which 53 sites on 37 proteins were defined as high-confidence sites (PSM Ն 2) (Fig. 3B and supplemental Table S2). GO term analyses revealed a significant enrichment of identified high-confidence proteins in the nucleus (p ϭ 2.03E-11) (Fig. 3D, and supplemental Table S7), which were consistent with previous studies in SUMO field.
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