Identification, Quantification, and Characterization of Glycopeptides in Reversed-Phase HPLC Separations of Glycoprotein Proteolytic Digests
✍ Scribed by J.S. Rohrer; G.A. Cooper; R.R. Townsend
- Publisher
- Elsevier Science
- Year
- 1993
- Tongue
- English
- Weight
- 852 KB
- Volume
- 212
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
Monosaccharide analysis by high (\mathrm{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 \mathrm{nmol}) ). This method, requiring no sample derivatization, identified four asparaginelinked ( (\mathbf{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 (\mathbf{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 (\mathrm{N})-acetylneuraminic acid (Neu5Ac). The (\mathbf{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. c 1993 Academic Press, Inc.