Antibody-free enrichment method for proteome-wide analysis of endogenous SUMOylation sites

Highlights: • The first antibody-free enrichment method for endogenous SUMOylation sites was proposed. • The specific enrichment of SUMOylated peptides was achieved by the strong anion exchange chromatography. • Asp-N/Glu-C were utilized to cut the enriched SUMOylated peptides into proper length for MS identification. • The simultaneous and unbiased identification of SUMO1/2/3-modified sites was achieved. SUMOylation is a reversible post-translational modification that plays crucial roles in numerous cellular processes. Although anti-SUMO antibodies have been applied to analyze exogenous and endogenous SUMOylation, such immunoprecipitation enrichment strategy is applicable only for the enrichment of one specific SUMO type in mammalian cells, unable to map the global landscape of all endogenous SUMOylation simultaneously. To address this issue, we proposed an antibody-free strategy to enrich and profile endogenous SUMO1/2/3-modified peptides simultaneously. Upon trypsin digestion, the SUMO1- and SUMO2/3-modified peptides contained SUMO remnants with 7 and 9 acidic amino acids respectively, which carried more negative charges at high pH and could interact with strong anion exchange (SAX) materials more strongly than non-SUMOylated peptides, thus enabling the specific enrichment of endogenous SUMOylated peptides. Followed by the secondary digestion with Asp-N/Glu-C to generate smaller SUMOylated peptides with proper length for MS identification, off-line high-pH C18 pre-fractionation and low pH nanoRPLC-ESI-MS/MS analysis, 177 SUMO1-modified sites and 74 SUMO2/3-modified sites were unbiasedly identified in HeLa cell lysate. To the best of our knowledge, this was the first antibody-free strategy to comprehensively profile various endogenous SUMOylation sites, demonstrating the great potential in the comprehensive analysis of endogenous SUMOylation across various species and organs, which might further facilitate the understanding of SUMO's function in physiology and pathology.