Figure 2

Site-specific analysis of heparan sulfate linkage region glycopeptides.

(a–d) Trypsin-digested perlecan was applied onto a SAX-column and enriched for GAG-glycopeptides. The resulting preparation was digested with heparinase and analyzed with nLC-MS/MS. (a) An extracted-ion current chromatogram of MS2-spectra which have been filtered for the presence of the m/z 362.11 diagnostic ion, representing the dehydrated disaccharide ion [ΔHexAGlcNAc]⁺, revealed several 362-related peaks eluting at various positions (b) An HS-glycopeptide (1363.48; 4+) was identified at 43.6 min and the MS2-spectrum (NCE 20%) showed characteristic glycosidic fragments. (c) Enlarged view of the low mass range (m/z 100–250) enabled the identification of several GlcNAc-derived oxonium ions. (d) MS2-fragmentation at higher energy (NCE 30%) provided several y-ions and one b-ion. The HS-glycopeptide is derived from the N-terminal domain of perlecan (d, insert). All ions are [M + zH]⁺ and their charges are shown as superscript when z > 1.