Of all posttranslational modifications known,
glycosaminoglycans (GAGs) remain one of the most challenging to study, and despite the recent years of advancement in MS technologies and bioinformatics, detailed knowledge about the complete structures of GAGs as part of
proteoglycans (PGs) is limited. To address this issue, we have developed a protocol to study PG-derived GAGs.
Chondroitin/
dermatan sulfate conjugates from the rat
insulinoma cell line, INS-1832/13, known to produce primarily the PG
chromogranin-A, were enriched by
anion-exchange chromatography after
pronase digestion. Following
benzonase and
hyaluronidase digestions, included in the sample preparation due to the apparent interference from
oligonucleotides and
hyaluronic acid in the analysis, the GAGs were orthogonally depolymerized and analyzed using nano-flow reversed-phase LC-MS/MS in negative mode. To facilitate the data interpretation, we applied an automated LC-MS peak detection and intensity measurement via the
Proteome Discoverer software. This approach effectively provided a detailed structural description of the nonreducing end, internal, and linkage region domains of the CS/DS of
chromogranin-A. The copolymeric CS/DS GAGs constituted primarily consecutive
glucuronic-acid-containing
disaccharide units, or CS motifs, of which the N-
acetylgalactosamine residues were 4-O-sulfated, interspersed by single
iduronic-acid-containing
disaccharide units. Our data suggest a certain heterogeneity of the GAGs due to the identification of not only CS/DS GAGs but also of GAGs entirely of CS character. The presented protocol allows for the detailed characterization of PG-derived GAGs, which may greatly increase the knowledge about GAG structures in general and eventually lead to better understanding of how GAG structures are related to biological functions.