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Proteome-wide Mapping of Endogenous SUMOylation Sites in Mouse Testis*

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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.

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Author contributions: L.C., J.T., C.D., and F.H. designed research; L.C., J.T., and L.S. performed research; L.C., L.S., Z.G., K.L., Y.W., and R.G. analyzed data; Y.L., F.Z., C.D., J.Q., and F.H. provided scientific support; L.C., C.D., and F.H. wrote and revised the manuscript.

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This work was supported by the following funds: Ministry of Science and Technology of China (2016YFA0502500), National Program on Key Basic Research Project (2014CBA02000 and 2013CB910802), National International Cooperation Grant (2014DFB30010, 2014DFB30020, 2012DFB30080 and CPRIT RP110784), National High-tech R&D Program of China (2015AA020108 and 2014AA020201), and Chinese State Key Project Specialized for Infectious Diseases (2012ZX10002012-006).

This article contains supplemental material.

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These authors contributed equally to this work.