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. Semi-quantitative data suggest that the glycosylation of hydroxylysine residues is site-specific.

Recombinant expression of hydroxylated human collagen in Escherichia coli

“…Hyp occurs exclusively at position y (Fietzek and Rauterberg 1975), recent studies showed that Hyp also occurs at position x in fibrillar collagens (Song and Mechref 2013;Weis et al 2010).…”

“…The presence of Hyp at both positions x and y by contrast, further stabilized the triple helical conformation of the peptides (Berisio et al 2004). The detection of several Hyp residues at position x on various types of collagen makes it difficult to predict the positional impact of Hyp on the thermal stability of more complex polypeptides (Bann and Bachinger 2000;Buechter et al 2003;Song and Mechref 2013). Although early work demonstrated that…”

Identification of Glycopeptides with Multiple Hydroxylysine O-Glycosylation Sites by Tandem Mass Spectrometry

“…38 A total of five MS/MS data sets were collected, containing 12 908 CID, 13 986 HCD, and 1 937 ETD MS/MS spectra. 7 …”

“…Following the preprocessing steps in Ref. 7, noise peaks were removed using a signal-to-noise ratio 1.5, and MS/MS spectra without peaks were removed.…”

“…[5][6][7] In bottom-up MS-based glycosylation analysis, glycoproteins are first digested into short glycopeptides by a protease, and then the resulting glycopeptides are enriched and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). 8,9 Fragmentation methods in MS/MS determine the patterns of fragment ions observed in MS/MS spectra of glycopeptides.…”