Characterization of endogenous SUMOylation sites by click chemistry-based proteomics.

SUMOylation, an essential ubiquitin-like modification in eukaryotes, plays vital roles in both physiological and pathological regulation, positioning it as a promising therapeutic target. However, the low abundance of SUMOylation and the high enzymatic activity of sentrin/SUMO-specific proteases (SENPs) complicate the identification of endogenous sites. In this study, we integrated click chemistry, acid cleavage, and SUMOylated peptide enrichment into the workflow and developed a promising methodology for system-wide identification of SUMOylation sites. In total, we identified 962 endogenous SUMOylation sites in HEK293T cells under heat shock conditions, which showed good complementarity with previous studies. Our approach uncovered 105 potentially new sites, including SSRP1-K248/K319/K612, DHX9-K806, and ILF3-K241, which showed high conservation and were located in functionally important domains. The overlap between SUMOylation sites and the known ubiquitination or acetylation sites suggested the potential PTM crosstalks. KEGG analysis further suggested SUMOylated proteins were associated with carbon metabolism and biosynthesis of amino acids pathways. Collectively, this study provides a valuable tool for systematically identifying SUMOylation sites, advancing further biological understanding of their dynamic regulatory networks and pathophysiological mechanisms.