LC-MS/MS identification of the o-glycosylation and hydroxylation of amino acid residues of collagen α-1 (II) chain from bovine cartilage

Ehwang Song, Yehia Mechref

Research output: Contribution to journalArticlepeer-review

24 Scopus citations

Abstract

O-Glycosylation of collagen is a unique type of posttranslational modifications (PTMs) involving the attachment of galactose (Gal) or glucose-galactose (Glc-Gal) moieties to hydroxylysine (HyK). Also, hydroxyproline (HyP) result from the posttranslational hydroxylation of some proline residues in collagen. Here, LC-MS/MS was effectively employed to identify 23 O-glycosylation sites and a large number of HyP residues associated with bovine type II collagen α-1 chain (CO2A1). The modifications of the 23 O-glycosylation sites varied qualitatively and quantitatively. Both Gal and Glc-Gal moieties occupied 22 of the identified glycosylation sites, while K773 was observed as unmodified. A large number of HyP residues at Yaa positions of Gly-Xaa-Yaa motif were detected. HyP residues at Xaa positions of Gly-HyP-HyP, Gly-HyP-Ala, and Gly-HyP-Val motifs were also observed. Notably, HyP residue of Gly-HyP-Gln motif was detected, which has not been previously reported. Moreover, the deamidation of 8 Asn residues was identified, of which 2 Asp residues were observed at different retention times because of isomerization (Asp vs isoAsp). Partial macroheterogeneities of some CO2A1 glycosylation sites were revealed by LC-MS/MS analysis. ETD experiments revealed partial macroheterogeneities associated with K299-K308, K452-K464, K464-K470, and K857-K884 glycosylation sites. Semiquantitative data suggest that the glycosylation of hydroxylysine residues is site-specific.

Original languageEnglish
Pages (from-to)3599-3609
Number of pages11
JournalJournal of Proteome Research
Volume12
Issue number8
DOIs
StatePublished - Aug 2 2013

Keywords

  • CO2A1
  • LC-MS/MS
  • glycopeptides
  • glycosylation
  • hydroxylation
  • quantitation
  • type II collagen

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