title: LC-MS/MS identification of the O-glycosylation and hydroxylation of amino acid residues of collagen α-1 (II) chain from bovine cartilage.
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.
authors.affiliation: Department of Chemistry and Biochemistry, Texas Tech University , Lubbock, Texas 79409, USA.
authors.forename: Ehwang
authors.initials: E
authors.lastname: Song
authors.forename: Yehia
authors.initials: Y
authors.lastname: Mechref
mesh_terms: D020816:Amino Acid Motifs; D000596:Amino Acids / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D024042:Collagen Type I / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006900:Hydroxylation; D006901:Hydroxylysine / Q000032:analysis*; D006909:Hydroxyproline / Q000032:analysis*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D053719:Tandem Mass Spectrometry; D014020:Tissue Extracts / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D024042:Collagen Type I; D014020:Tissue Extracts; C006931:bovine cartilage extract; D006901:Hydroxylysine; D006909:Hydroxyproline