Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Jun;11(6):M111.010397.
doi: 10.1074/mcp.M111.010397. Epub 2012 Jan 13.

Development of a novel method for analyzing collagen O-glycosylations by hydrazide chemistry

Yuki Taga  1 , Masashi Kusubata, Kiyoko Ogawa-Goto, Shunji Hattori
Affiliations

Development of a novel method for analyzing collagen O-glycosylations by hydrazide chemistry

Yuki Taga et al. Mol Cell Proteomics. 2012 Jun.

Abstract

In recent years, glycopeptide purification by hydrazide chemistry has become popular in structural studies of glycoconjugates; however, applications of this method have been almost completely restricted to analysis of the N-glycoproteome. Here we report a novel method for analyzing O-glycosylations unique to collagen, which are attached to hydroxylysine and include galactosyl-hydroxylysine and glucosyl-galactosyl-hydroxylysine. We established a hydrazide chemistry-based glycopeptide purification method using (1) galactose oxidase to introduce an aldehyde into glycopeptides and (2) formic acid with heating to elute the bound glycopeptides by cleaving the hydrazone bond. This method allows not only identification of O-glycosylation sites in collagen but also concurrent discrimination of two types of carbohydrate substitutions. In bovine type I and type II collagens, galactosyl-hydroxylysine /glucosyl-galactosyl-hydroxylysine -containing peptides were specifically detected on subsequent comprehensive liquid chromatography (LC)/MS analysis, and many O-glycosylation sites, including unreported ones, were identified. The position of glycosylated hydroxylysine, which is determined by our unambiguous and simple method, could provide insight into the physiological role of the modifications.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Schematic diagram of the hydrazide method for collagen O-glycosylations. A, Purification strategy for standard of hydroxylysine glycosides and collagen. Purified GHL/GGHL and collagen samples, which were denatured and then digested by trypsin, were oxidized by galactose oxidase. The oxidized samples were coupled to hydrazide resin under adjusted weak acidic conditions, and unbound/nonspecifically bound compounds were removed by washing. The bound compounds were released from hydrazide resin by heating with formic acid and were analyzed by LC/MS after reduction treatment. B, Details of the chemical reactions of GGHL using the hydrazide method. The hydroxyl group at the C6 position of galactose was oxidized to the aldehyde group by galactose oxidase (1). The aldehyde group was coupled to hydrazide resin by forming a hydrazone bond (2). The captured GGHL was eluted by heated 0.1% formic acid (3).
Fig. 2.
Fig. 2.
Oxidation efficiency and recovery rate of GHL/GGHL standards using the hydrazide method. One sample was oxidized by galactose oxidase only and the other was oxidized by the enzyme with the coordinated addition of catalase and HRP. The oxidized samples were coupled to hydrazide resin and released by heating under acidic conditions following resin washes. The samples (preoxidation, postoxidation (oxidant), those unbound to hydrazide resin (flow-through), and postelution (eluant)) were reduced with sodium borodeuteride. 13C6 15N2-lysine was added as an internal standard, and then the samples were subjected to MRM analysis. The relative amounts of deuterium-labeled oxidation products of GHL/GGHL of each sample were calculated by comparing them to the original GHL/GGHL of preoxidation. The data represent the mean ± standard deviation of five separate experiments.
Fig. 3.
Fig. 3.
Identification of GHL/GGHL peptides of type II collagen. A, TIC of type II collagen. B, Survey MS spectrum obtained at 14.76 min in the TIC. The m/z 641.0 (z = 3+) and 961.0 (z = 2+) ions were both derived from the same GHL peptide, and the 695.0 (z = 3+) and 1042.0 (z = 2+) ions were derived from the identical peptide with GGHL instead of GHL. The labeling numbers represent monoisotopic masses. C, MS/MS spectrum of the precursor ions at m/z 641.0 (z = 3+) and D, m/z 695.0 (z = 3+) selected from the survey MS scan at 14.76 min. The m/z 641.0 ion was identified as GFOGQDGLAGPK*GAOGER (O indicates hydroxyproline and K* indicates GHL), and the m/z 695.0 ion was identified as GFOGQDGLAGPK#GAOGER (K# indicates GGHL). -G represents deglycosylated fragment ions (-162) of GHL peptide, and -GG represents deglycosyated fragment ions (-324) of GGHL peptide. The extracted ion chromatograms of the GHL peptide (m/z 640.96–641.29) and GGHL peptide (m/z 694.98–695.31) were also shown. E, MALDI-TOF mass spectrum of the fraction that contained the peaks shown in Fig. 3B. The peaks at m/z 1922.95 and m/z 2085.04 correspond to peptides GFOGQDGLAGPK*GAOGER and GFOGQDGLAGPK#GAOGER, respectively. F, The result of the N-terminal amino acid sequence analysis of the fraction collected from LC/MS analysis at ∼14.76 min. It was determined as GFOGQDGLAGP(X)GAOGER (X indicates blank). *The repetitive yield of hydroxyproline was not calculated because it divided into two peaks and phenylthiohydantoin-hydroxyproline was not available commercially.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Spiro R. G. (1967) The structure of the disaccharide unit of the renal glomerular basement membrane. J. Biol. Chem. 242, 4813–4823 - PubMed
    1. Shinkai H., Yonemasu K. (1979) Hydroxylysine-linked glycosides of human complement subcomponent C1q and various collagens. Biochem. J. 177, 847–852 - PMC - PubMed
    1. Wang C., Luosujärvi H., Heikkinen J., Risteli M., Uitto L., Myllylä R. (2002) The third activity for lysyl hydroxylase 3: galactosylation of hydroxylysyl residues in collagens in vitro. Matrix Biol 21, 559–566 - PubMed
    1. Schegg B., Hülsmeier A. J., Rutschmann C., Maag C., Hennet T. (2009) Core glycosylation of collagen is initiated by two beta(1-O)galactosyltransferases. Mol. Cell Biol. 29, 943–952 - PMC - PubMed
    1. Kivirikko K. I., Myllylä R. (1979) Collagen glycosyltransferases. Int Rev Connect Tissue Res 8, 23–72 - PubMed

MeSH terms

LinkOut - more resources