Fig 1: Differential proteolytic processing of glycoproteins in samples from wt and COSMC ko cells. (a) Glycoproteins, as marked in Figure 3a with a log2 (ko/wt) abundance ratio <0.2, were extracted and correlated (as indicated by lines) with (b) their respective identified neo‐N termini, which showed at least a 20% increase in abundance in samples from ko compared to wt cells. Time‐series samples were treated as replicates for genotype quantitative comparisons. “Position” indicates the position of the neo‐N terminus based on the UniProt fasta sequences. (c) Correlation of identified neo‐N termini with glycopeptides extracted from Steentoft et al. highlighted cleavages near annotated glycosites (in red, “?” denotes for unassigned sites) in DKK1 (O94907), FN1 (P02751), CSF (P09603), CD74 (P04233), and PTPRG (P23470) proteins.
Fig 2: Validation of CTSS‐mediated cleavage of CD74 via PRM. (a) Recombinant human CD74 proteins in both glycosylated (produced in HEK cells) and non‐glycosylated (produced in E. coli) forms were treated with activated recombinant human CTSS. Control samples for each protein type without CTSS were included. The presence of the semi‐tryptic peptide VTSQNLQLENLR was quantified using PRM. The bar graph shows the total ion current (TIC) normalized area‐under‐the‐curve (AUC) for this specific peptide across the samples (n = 2), presented as mean ± SD. (b) Glycoproteomics profiling of the CTSS cleavage site‐containing LDKLTVTSQNLQLENLR peptide on the recombinant human protein CD74. The bar graph displays glycosylation at three distinct sites Ser85, Thr82, and Thr84 (highlighted in red)—on the peptide, showing the specific locations and the frequency of glycan attachments. (c) Glycoproteomics profiling of the CTSS cleavage site‐containing LDKLTVTSQNLQLENLR peptide reveals the variety of glycan compositions found within the peptide's sequence. Hex, hexose; HexNAc, N‐acetylhexosamine; Fuc, fucose; NeuAc, N‐acetylneuraminic acid.
Supplier Page from Abcam for Recombinant Human CD74 protein (His tag N-Terminus)