Fig. 1.

Graphic depiction of the gene engineering strategy and glycoproteomics workflow. A, Evaluation of expressed repertoire of GALNTs in HEK293 cells assessed by RNAseq analysis (top) and enzyme protein expression by ICC with mAbs (below). B, Depiction of the biosynthetic pathway of core1 O-glycosylation and the genes targeted for generation of isogenic HEK293^SC and HEK293^WT with KO of GALNT1, T2, T3, T7, T10, or T11. C, Depiction of the quantitative glycoproteomics workflow using dimethyl labeling. Trypsin digests from total cell lysates of paired isogenic HEK293^SC and HEK293^SCΔT were labeled with “light” (L) and “medium” (M) isotopes, respectively, mixed and GalNAc-glycopeptides enriched on a long VVA column, and Labeled glycopeptides were analyzed by nLC-MS/MS.