Abstract
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.
Highlights
Protocol developed to structurally characterize glycosaminoglycans of proteoglycans. Comprehensive characterization of cellular glycosaminoglycan structures. Relative quantification of nonreducing end, internal, and linkage region domains. Overall chondroitin/dermatan sulfate glycosaminoglycan structures of chromogranin-A
When developing a glycomics approach for domain mapping of PG-derived GAGs, the analytical method needs to be taken into consideration, and the sample preparation
Instead of trypsin digestion, which is suitable for proteomics and glycoproteomics where peptide sequences of 5–30 amino acids are desirable [21], extensive digestion with pronase, a nonspecific protease, was performed to more completely hydrolyze the proteins to reduce the number of peptide variants, increase the analytical sensitivity, and facilitate the data interpretation of the linkage region products
Summary
In Brief Glycosaminoglycans (GAGs) remain one of the most challenging posttranslational modifications to study, much due to their structural complexity and heterogeneity, and new methods for analysis are required. 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 anionexchange chromatography after pronase digestion. 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 secretory pattern of the INS-1832/13 cells allowed us to conclude an overall CS/DS GAG structure of CgA
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
