Comparative glycoproteomics of embryonic stem cells identifies new players in ricin toxicity

Rapid advances in the fields of DNA-, RNA-, polypeptide-sequencing and metabolomics have markedly enhanced our understanding of fundamental biological processes. In contrast to other post-translational modifications (PTMs), the most abundant PTM, glycosylation, remains largely unexplored at the proteome scale because of a scarcity of technologies for profiling the immensely complex glycoproteome. We developed a novel quantitative approach for the identification of intact glycopeptides from comparative proteomic data-sets, allowing us to not only identify complex sugar structures but also to directly map them to the corresponding glycosylation sites in the associated proteins at the proteome scale. Applying this method to human and murine embryonic stem cells, we were able to nearly double the number of all experimentally confirmed glycoproteins, including the identification of completely unknown glycosylation sites, multiple glycosylated stemness factors and the elucidation of evolutionary conserved core as well as species-specific glycoproteins in embryonic stem cells. Specificity of our method was confirmed in sister stem cells carrying repairable mutations in enzymes required for fucosylation, Fut9 and Slc35c1. Since ablation of fucosylation confers cellular resistance towards the bioweapon ricin (see accompanying paper), our method also allowed us to directly identify the proteins that carry the terminal fucosylation code for ricin toxicity; genetic mutations in these proteins functionally confirmed their role in ricin killing. This novel comparative and high-throughput glycoproteomics platform should allow for entirely novel insights in protein glycosylation and sugar modifications involved in nearly all biological systems.