biostudies-literatureUnknownvan Gerwen JWellcome TrustNIGMS NIH HHSBiotechnology and Biological Sciences Research CouncilProtein ubiquitination regulates cell biology through diverse avenues, from quality control-linked protein degradation to signaling functions such as modulating protein-protein interactions and enzyme activation. Mass spectrometry-based proteomics has allowed proteome-scale quantification of hundreds of thousands of ubiquitination sites (ubi-sites), however the functional importance and regulatory roles of most ubi-sites remain undefined. Here, we assembled a human reference ubiquitinome of 108,341 ubi-sites by harmonizing public proteomics data. We identified a core subset of ubi-sites under evolutionary constraint through alignment of ubiquitin proteomics data from six non-human species, and determined ultra-conserved ubi-sites recurring at regulatory hotspots within protein domains. PerbioRxiv : the preprint server for biology2025.10.08.681129https://www.ebi.ac.uk/biostudies/studies/S-EPMC12632403biostudies-literatureThe functional landscape of the human ubiquitinome.PMC12632403223745/Z/21/ZR35 GM148339BB/Y513829/1DP2 GM119132R01 GM136994BB/S017054/1Studer RLang KFottner MCarrano ACTroemel ERvan Gerwen JBeltrao PBakowski MABennett EJHarper JWStrumillo MWang SMartin MJJones ARBarrio-Hernandez ISchnacke PBusby BVizcaino JABoswell EfalseThe functional landscape of the human ubiquitinome.Protein ubiquitination regulates cell biology through diverse avenues, from quality control-linked protein degradation to signaling functions such as modulating protein-protein interactions and enzyme activation. Mass spectrometry-based proteomics has allowed proteome-scale quantification of hundreds of thousands of ubiquitination sites (ubi-sites), however the functional importance and regulatory roles of most ubi-sites remain undefined. Here, we assembled a human reference ubiquitinome of 108,341 ubi-sites by harmonizing public proteomics data. We identified a core subset of ubi-sites under evolutionary constraint through alignment of ubiquitin proteomics data from six non-human species, and determined ultra-conserved ubi-sites recurring at regulatory hotspots within protein domains. Per2025-01-01T00:00:00Z2025 Oct2026-07-02T03:14:15.756Z2026-07-02T03:08:54.852ZS-EPMC126324034127905110.1101/2025.10.08.681129