Comparative glycoproteomics of stem cells identifies new players in ricin toxicity

Abstract

Glycosylation, the covalent attachment of carbohydrate structures onto proteins, is the most abundant post-translational modification. Over 50% of human proteins are glycosylated, which alters their activities in diverse fundamental biological processes. Despite the importance of glycosylation in biology, 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, and we discover proteins that carry a fucosylation-dependent sugar code for ricin toxicity. Mutations disrupting a subset of these proteins render cells ricin resistant, revealing new players that orchestrate ricin toxicity. Our comparative glycoproteomics platform, SugarQb, enables genome-wide insights into protein glycosylation and glycan modifications in complex biological systems.

Global and site‐specific analysis of protein glycosylation in complex biological systems with Mass Spectrometry

“…Glycoproteins in human cell lines and stem cells have been extensively studied (Alvarez‐Manilla et al, ; Dai et al, ; Boheler et al, ; Bausch‐Fluck et al, ; Konze et al, ; Li et al, ; Stadlmann et al, ), which provide much valuable information. Glycoproteomic analysis has advanced from glycoprotein identification‐centric studies to site‐specific investigation, and glycan structural characterization of intact glycopeptides and glycoproteins.…”

The Cannabis Multi-Omics Draft Map Project

“…Files were filtered at the PSM level for lysine acetylation and phosphorylation of serine/threonine, respectively. To search for the presence of glycosylated peptides, the SugarQb program 24,25 was used in Proteome Discoverer 2.1 using the default workflow as described in the instruction manual on pd-nodes.org. For this analysis only one fractionated file set was searched, containing the combined flower samples 7-12 that were high pH fractionated as described above.…”

Analysis of PNGase F‐Resistant N‐Glycopeptides Using SugarQb for Proteome Discoverer 2.1 Reveals Cryptic Substrate Specificities

“…The precursor mass tolerance was set to 10 ppm, the fragment mass tolerance was set to 25 mmu. Amino acid sequence identification was based on matching singly‐charged b‐ and y‐fragment ion series, considering ammonia and water losses, as well as the neutral loss of HexNAc . The resulting peptide spectrum matches (PSMs) were manually prefiltered (i.e., best scoring search engine rank 1 PSMs only, peptide length greater than six amino acids), sorted by the respective search engine score value and then filtered to 1% FDR using the concatenated forward and decoy approach .…”

“…[8,9] As these long-standing observations suggest important technical limitations to the comprehensive characterization of the N-glycoproteome in PNGase F-dependent workflows, we were prompted to evaluate their impact on the analysis of the N-glycoproteome by identifying and analyzing PNGase F-resistant N-glycopeptides using the recently developed SugarQb platform. [11] Aiming at a comprehensive characterization of intact glycopeptides from complex samples, we recently developed a collection of data interpretation tools, which allows for the automated identification of intact glycopeptides from high-resolution HCD MS/MS datasets, using well-established proteomic MS/MS search engines (e.g., MASCOT, SEQUEST-HT, MS Amanda [12] ). SugarQb (www.imba.oeaw.ac.at/sugarqb) analyses MS/MS spectra for the presence of potential [peptide + HexNAc]+ fragment ions.…”

“…To more precisely quantify the susceptibility of the N-glyco proteome to PNGase F-treatment, we used a comparative glycoproteomic approach. [11] For this, we labeled tryptic digests of mESC whole cell lysates with TMT-6plex (Thermo; 200 mg of tryptic peptides per TMT channel), desalted and treated them with increasing amounts of PNGase F (i.e., 0, 0.5 U and 5 U PNGase F mg −1 protein in 200 mM Tris/HCl, pH 8.0) for 18 h at 37°C. After incubation, the individual samples were adjusted to pH 2 by the addition of 10% formic acid, pooled and desalted using SPE C18 cartridges.…”