Optimization of data acquisition and antibody enrichment conditions.A, total HexNAc-containing PSMs across different HCD collision energies (CEs) (300 μg of mESC WCL). Both HCD and product triggered EthcD (pd-EthcD) PSMs are shown. Only HCD CEs were altered. B, distribution of PEP2D scores for HexNAc-containing PSMs separated by dissociation method. Only HCD CEs were altered. EThcD supplemental activation energy was held constant at 25%. C, number of PSMs per charge state across different HCD CEs. D, relative HexNAc-containing PSMs titrating down the amount of antibody from the recommended levels. Values are relative to the 1× experiment. E, O-HexNAc glycopeptide specificity for decreasing amounts of antibody. Specificity was calculated by dividing the number of HexNAc-containing PSMs by the total number of PSMs. F, overlap in distinct glycoprotein identifications from triplicate 1 mg mESC WCL enrichments. G, overlap in distinct glycopeptide identifications from three separate enrichments from 1 mg aliquots of digested mESC WCL digests.