title: Identification, quantification, and characterization of glycopeptides in reversed-phase HPLC separations of glycoprotein proteolytic digests.
abstract: Monosaccharide analysis by high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC/PAD) was used to identify the glycopeptides in the reversed-phase (RP-HPLC) separation of a bovine fetuin tryptic digest (1.6 nmol). This method, requiring no sample derivatization, identified four asparagine-linked (N-linked) glycopeptides and at least seven serine/threonine-linked (O-linked) glycopeptides. Glycopeptide identification was confirmed by Edman sequencing. Monosaccharide quantification of each glycopeptide suggested that all of the N-linked glycopeptides were the complex type and all the O-linked glycopeptides were sialylated. We determined that glycopeptides could be prepared by acidic reversed-phase chromatography with less than 3% loss of N-acetylneuraminic acid (Neu5Ac). The N-linked glycopeptides of bovine fetuin were prepared, digested with N-glycosidase F (PNGase F), and their oligosaccharides analyzed by HPAEC/PAD. These oligosaccharide profiles revealed that the Asn-138 oligosaccharide attachment site contained the majority of the disialylated and monosialylated oligosaccharides. The Asn-158 oligosaccharide attachment site contained the majority of the tetrasialylated oligosaccharides.
authors.affiliation: Dionex Corporation, Sunnyvale, California 94086.
authors.forename: J S
authors.initials: JS
authors.lastname: Rohrer
authors.forename: G A
authors.initials: GA
authors.lastname: Cooper
authors.forename: R R
authors.initials: RR
authors.lastname: Townsend
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange; D006020:Glycopeptides / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D006031:Glycosylation; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000032:analysis; D009844:Oligosaccharides / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D014357:Trypsin; D000509:alpha-Fetoproteins / Q000302:isolation & purification
chemical_list: D006020:Glycopeptides; D009005:Monosaccharides; D009844:Oligosaccharides; D010446:Peptide Fragments; D000509:alpha-Fetoproteins; D014357:Trypsin