Characterization of Glycan Structures of Chondroitin Sulfate-Glycopeptides Facilitated by Sodium Ion-Pairing and Positive Mode LC-MS/MS

Abstract

Purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS) characterization of glycopeptides, originating from protease digests of glycoproteins, enables site-specific analysis of protein N- and O-glycosylations. We have described a protocol to enrich, hydrolyze by chondroitinase ABC, and characterize chondroitin sulfate-containing glycopeptides (CS-glycopeptides) using positive mode LC-MS/MS. The CS-glycopeptides, originating from the Bikunin proteoglycan of human urine samples, had ΔHexAGalNAcGlcAGalGalXyl-O-Ser hexasaccharide structure and were further substituted with 0-3 sulfate and 0-1 phosphate groups. However, it was not possible to exactly pinpoint sulfate attachment residues, for protonated precursors, due to extensive fragmentation of sulfate groups using high-energy collision induced dissociation (HCD). To circumvent the well-recognized sulfate instability, we now introduced Na+ ions to form sodiated precursors, which protected sulfate groups from decomposition and facilitated the assignment of sulfate modifications. Sulfate groups were pinpointed to both Gal residues and to the GalNAc of the hexasaccharide structure. The intensities of protonated and sodiated saccharide oxonium ions were very prominent in the HCD-MS2 spectra, which provided complementary structural analysis of sulfate substituents of CS-glycopeptides. We have demonstrated a considerable heterogeneity of the bikunin CS linkage region. The realization of these structural variants should be beneficial in studies aimed at investigating the importance of the CS linkage region with regards to the biosynthesis of CS and potential interactions to CS binding proteins. Also, the combined use of protonated and sodiated precursors for positive mode HCD fragmentation analysis will likely become useful for additional classes of sulfated glycopeptides. Graphical Abstractᅟ Electronic supplementary materialThe online version of this article (doi:10.1007/s13361-016-1539-1) contains supplementary material, which is available to authorized users.

Deep Sequencing of Complex Proteoglycans: A Novel Strategy for High Coverage and Site-specific Identification of Glycosaminoglycan-linked Peptides

“…Until the present, much of the knowledge of tissue proteoglycan glycosylation has derived from classical biochemical and antibody-binding studies. To date, glycoproteomics methods have been used to characterize bikunin (16), CSPGs from urine and spinal fluid (28,30), C. elegans (71), and the CS/HS PG perlecan (29). We report glycoproteomics methods to assess site-specific glycosylation of the SLRP decorin and the hyalectans aggrecan, brevican and neurocan.…”

Proteomics, Glycomics, and Glycoproteomics of Matrisome Molecules

“…Such linker glycopeptides can be identified by the presence of a diagnostic oxonium ion for CS and HS proteoglycans (146). The linker saccharide glycopeptides detected for CSPGs were modified with sulfate, phosphate, fucose and/or sialic acid (147)(148)(149). This approach has been used to analyze PGs from biological fluids including urine and cerebrospinal fluid (149).…”

Expanding the Chondroitin Sulfate Glycoproteome — But How Far?

“…Whether patients with B3GALT6 mutations also have increased levels of the non-canonical trisaccharide linkage region of bikunin in their urine remains to be determined. Taken together, at least 14 different variants of the bikunin linkage region (including canonical and non-canonical sequences) have now been described, thus demonstrating a much greater variability than previously appreciated (Gomez Noborn et al, 2015;Nilsson et al, 2017;Persson et al, 2019). Xylose phosphorylation has been shown to function as a molecular switch to regulate the proteoglycan biosynthesis (Wen et al, 2014).…”

“…However, the reports have been limited to only a few separate experimental systems and information on potential combinations of linkage region modifications is still scarce. To this aim, our glycoproteomic analysis of human bikunin and other proteoglycans from urine, CSF, and plasma have revealed an unexpected linkage region complexity, with different combinations of sulfation, phosphorylation and sialylation ( Gomez Toledo et al, 2015 ; Noborn et al, 2015 ; Nasir et al, 2016 ; Nilsson et al, 2017 ). Furthermore, the structural variability of human bikunin was further increased by large variations in the mucin type O -linked glycosylation found nearby the CS site ( Gomez Toledo et al, 2015 ).…”