Characterization of sensitivity and specificity of anti-O-GlcNAc antibodies and their comparison with WGA.A, total number of HexNAc-containing PSMs obtained by enrichment with Ab (blue bars) or WGA (green bars) across a range of input levels of mESC WCL digest; the optimal amount of Ab determined in Figure 1 was used. B, numbers of HexNAc-containing PSMs obtained at varying input levels and separated by dissociation method. “pd-EThcD” indicates the EThcD scan was triggered by detection of HexNAc fragment ions in the preceding HCD scan. C, extracted ion chromatograms (XICs) from the WGA-based glycopeptide enrichment experiments of HexNAc fragment ions (+/− 20 ppm of calculated masses) diagnostic for respective carbohydrates; 204.087 m/z represents HexNAc (black), 366.140 m/z for HexHexNAc (burgundy), and 274.092 m/z for sialic acid minus water (green). Base peak intensities are shown on the upper left. D, same as in (C), above, but for the anti-O-GlcNAc Ab-based glycopeptide enrichments. E, number of MS2 spectra containing one of the aforementioned HexNAc fragment ions from the respective enrichment strategies from 1 mg of peptide inputs. F, percentage of all MS2 scans with aforementioned HexNAc fragment ions from the respective enrichment strategies from 1 mg.