title: Development of a novel method for analyzing collagen O-glycosylations by hydrazide chemistry.
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.
authors.affiliation: Nippi Research Institute of Biomatrix, Toride, Ibaraki 302-0017, Japan. y-taga@nippi-inc.co.jp
authors.forename: Yuki
authors.initials: Y
authors.lastname: Taga
authors.forename: Masashi
authors.initials: M
authors.lastname: Kusubata
authors.forename: Kiyoko
authors.initials: K
authors.lastname: Ogawa-Goto
authors.forename: Shunji
authors.initials: S
authors.lastname: Hattori
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000592:standards; D003094:Collagen; D024042:Collagen Type I / Q000737:chemistry / Q000302:isolation & purification*; D024043:Collagen Type II / Q000737:chemistry / Q000302:isolation & purification*; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification*; D006031:Glycosylation; D006834:Hydrazines / Q000737:chemistry*; D006901:Hydroxylysine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D053719:Tandem Mass Spectrometry
chemical_list: D024042:Collagen Type I; D024043:Collagen Type II; D006023:Glycoproteins; D006834:Hydrazines; D010446:Peptide Fragments; D006901:Hydroxylysine; C001295:glucosylgalactosylhydroxylysine; C007817:galactosylhydroxylysine; D003094:Collagen