Comparative glycoproteomics of stem cells identifies new players in ricin toxicity

A novel quantitative approach to identify intact glycopeptides from comparative proteomic data sets, allowing inference of complex glycan structures and direct mapping of them to sites within the associated proteins at the proteome scale. Pinpointing glycoproteins The activity of more than half of human proteins is modified by their attachment to carbohydrate structures. To improve the identification and functional validation of such protein glycosylation, Josef Penninger and colleagues have developed a proteomics-based approach to identifying intact glycopeptides. The technique has high specificity and can pinpoint the location within the proteins where the glycosyl group has attached. The authors used their approach on human and mouse embryonic stem cells and identified nearly twice as many glycoproteins than were previously known. This analysis additionally led to the identification of several glycosylated 'stemness' factors, as well as of evolutionarily conserved and species-specific glycoproteins. The team also used the method to provide molecular insights into the toxicity of the bioweapon ricin. Glycosylation, the covalent attachment of carbohydrate structures onto proteins, is the most abundant post-translational modification 1 . Over 50% of human proteins are glycosylated, which alters their activities in diverse fundamental biological processes 2 , 3 . Despite the importance of glycosylation in biology 4 , the identification and functional validation of complex glycoproteins has remained largely unexplored. Here we develop a novel quantitative approach to identify intact glycopeptides from comparative proteomic data sets, allowing us not only to infer complex glycan structures but also to directly map them to sites within the associated proteins at the proteome scale. We apply this method to human and mouse embryonic stem cells to illuminate the stem cell glycoproteome. This analysis nearly doubles the number of experimentally confirmed glycoproteins, identifies previously unknown glycosylation sites and multiple glycosylated stemness factors, and uncovers evolutionarily conserved as well as species-specific glycoproteins in embryonic stem cells. The specificity of our method is confirmed using sister stem cells carrying repairable mutations in enzymes required for fucosylation, Fut9 and Slc35c1. Ablation of fucosylation confers resistance to the bioweapon ricin 5 , 6 , and we discover proteins that carry a fucosylation-dependent sugar code for r