Mapping O-GlcNAc sites and site-specific quantitation. A: Scheme for enrichment of O-GlcNAc peptides. B: Structure (inset) and CAD fragmentation of fully tagged O-GlcNAc peptide (YSPgTSPSK). [M+GlcNAc+GalNAz+Biotin+3H]3+ = 614.6, [M+H]+ = 866.5, [M+GlcNAc+H]+ = 1069.6. C: Flow chart for comparing site-specific O-GlcNAc RORs. Inset: Scheme for solid-phase BEMAD. D: Protein expression level dynamics in diabetic erythrocytes compared with normal erythrocytes. E: Specificity control for the enrichment and site-mapping. Samples were untreated or treated with hexosaminidase at 37°C for
48 h before going through the work flow. Base peak chromatograms are shown. NL, intensity in counts normalized to 1 s; TBTA,
Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine; TCEP, Tris(2-carboxyethyl)phosphine.