A Landscape Analysis of Human SUMOylation

SUMOylation is an understudied post-translational modification (PTM) linked to diverse physiological and pathological processes. Advances in enrichment strategies, combined with mass spectrometry (MS), have enabled large-scale mapping of SUMOylation sites. Here, we reanalyzed publicly available MS-based datasets to construct a comprehensive and high-confidence reference set of human SUMOylation sites. Our workflow integrated database searching and scoring through the Trans-Proteomic Pipeline (TPP) with a statistical approach to independently estimate global false localization rate (FLR) of modification sites. SUMOylated lysine sites identified at <5% FLR were classified into three confidence tiers and compared with a high-quality set of non-SUMOylated lysines. The Human SUMO Build comprises 35,721 SUMOylation sites across 6146 proteins. SUMOylated lysines were enriched within intrinsically disordered regions and underrepresented in tightly packed structural elements. These sites exhibited a higher frequency of nearby phosphosites at -2, +1, and +5, and were enriched for disease-linked variants at the modified lysine and the -2 position. Motif analysis revealed canonical, inverted, and novel SUMOylation motifs with flanking amino acid enrichment, including aspartic acid at -2, isoleucine and valine at -1, proline at +1, and glutamic acid at +2. Comparative analysis of SUMOylation and ubiquitination sites revealed that the two modifications frequently target the same lysine residue. Sites exclusively SUMOylated are preferentially located within intrinsically disordered regions, whereas sites exclusively ubiquitinated are enriched in secondary structural elements. Sites modified by both PTMs are enriched more strongly for disease-associated variants at the modified lysine and the -1 position than sites unique to SUMOylation or ubiquitination. Gene Ontology enrichment analysis linked motifs to biological processes, with most motifs contributing to chromatin remodeling, histone modification, and mRNA processing. The Human SUMO Build is publicly available through the PTMeXchange initiative, with data deposited in PRIDE and integrated into UniProtKB and PeptideAtlas to facilitate downstream analyses and predictive modeling.