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Identification of an attachment site (Ser-433) in rat chromogranin-A that carries either a CS or a HS chain. (A–B) Extracted ion chromatograms (XICs) of chondroitinase ABC and heparinases (II and III) treated INS-1 832/13 cell culture media. The XICs are filtered for the presence of CS (A) (m/z 362.10–362.12) and HS (B) (m/z 173.04–173.05) diagnostic ions, respectively. Of note, the peptide contains two missed cleavage sites and is thus the result of incomplete trypsin digestion (Note 11). (C–D) MS² spectra of CS and HS glycopeptides derived from the same peptide sequence of rat chromogranin-A. The chondroitinase ABC depolymerization generates residual CS hexasaccharides whereas the heparinase depolymerization generates residual HS tetrasaccharides. The spectra are collected at fragmentation energy of NCE 20% to provide abundant glycosidic fragmentation. The spectrum (C) displays a mass shift of 79.9567 u (difference between m/z = 362.1079; 1+ and m/z 442.0646; and 1+) demonstrating the presence of a sulfate group on the GalNAc residue. In (D) a mass shift of 212.0154 u was observed between m/z 575.5930; 3+ and m/z 646.2648; and 3+, demonstrating the presence of a xylose and a phosphate group (132.0423 u + 79.9663 u = 212.0086 u) as opposed to a xylose and a sulfate group (132.0423 u + 79.9568 u = 211.9991 u). (E–F) MS² spectra of the same CS and HS glycopeptides collected at NCE 35%. This higher fragmentation energy provides abundant peptide backbone b- and y-ions.