Ph 3 10 strips

LWAC isolation of Tn and T‐O‐glycopeptides was performed as described previously 27, 30. Briefly, proteins were reduced (5 mM DTT 45 min 60°C) and alkylated (10 mM IAA 30 min RT), digested with trypsin (Roche), and neuraminidase‐treated to remove sialic acid residues. For experiments in COSMC KO (SC), these steps were followed by labeling with light or medium isotopomeric dimethyl labels 42. The labeled digests were mixed in a 1:1 ratio in the following sets: HaCaT^SC (light) and HaCaT^SCΔT1 (medium), HaCaT^SC (light) and HaCaT^SCΔT2 (medium), and HaCaT^SC (light) and HaCaT^SCΔT3 (medium). The mixed pairs of digests were enriched using agarose‐bound VVA LWAC and eluted with GalNAc. For experiments in WT, digests of three clones of each ΔT1, ΔT2, and ΔT3, and one WT were labeled using the TMT‐10plex Kit (Thermo Scientific) according to the manufacturer's instructions. Labeled digests were mixed in an equimolar ratio and enriched using Jacalin LWAC, followed by elution with D‐galactose. Isoelectric focusing was performed on VVA‐enriched glycopeptides by a 3100 OFFGEL fractionator (Agilent) using pH 3–10 strips (GE Healthcare) 79. High pH fractionation was performed on TMT‐labeled Jacalin‐enriched glycopeptides, as well as peptides before enrichment. All glycopeptide samples were desalted by self‐made Stage Tips (C18 sorbent from Empore 3 M) and submitted to LC‐MS and HCD/ETD‐MS/MS.