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

Artikel i vetenskaplig tidskrift
Författare Jonas Nilsson
Fredrik Noborn
Alejandro Gomez Toledo
Waqas Nasir
Carina Sihlbom
Göran Larson
Publicerad i Journal of the American Society for Mass Spectrometry
Volym 28
Nummer/häfte 2
Sidor 229-41
ISSN 1879-1123
Publiceringsår 2017
Publicerad vid Institutionen för biomedicin, avdelningen för klinisk kemi och transfusionsmedicin
Core Facilities, Proteomics
Sidor 229-41
Språk en
Länkar dx.doi.org/10.1007/s13361-016-1539-...
www.ncbi.nlm.nih.gov/entrez/query.f...
Ämneskategorier Klinisk laboratoriemedicin

Sammanfattning

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 Abstract ᅟ.

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http://www.gu.se/forskning/publikation/?publicationId=246243
Utskriftsdatum: 2019-09-23