title: Sequential analysis of N- and O-linked glycosylation of 2D-PAGE separated glycoproteins.
abstract: A robust method has been developed that allows analysis of both N- and O-linked oligosaccharides released from glycoproteins separated using 2D-PAGE and then electroblotted to PVDF membrane. This analysis provides efficient oligosaccharide profiling applicable to glycoproteomic analysis. The method involves the enzymatic release of N-linked oligosaccharides using PNGase F followed by the chemical release of O-linked oligosaccharides using reductive beta-elimination and analysis using LC-ESI-MS. Oligosaccharides from the major plasma glycoproteins with a pI between 4 and 7 were characterized from the glycoforms of haptoglobin, alpha2-HS-glycoprotein, serotransferrin, alpha1-antitrypsin, and alpha1-antichymotrypsin. It was shown that the separation of protein glycoforms evident in 2D-PAGE is partially due to the combined sialylation of the O-linked and N-linked oligosaccharides. Bi-, tri- and tetra-antennary N-linked structures, which had differing levels of sialylation and fucosylation, were found to be present on the glycoproteins analyzed, together with O-linked oligosaccharides such as mono-, and disialylated T-antigen and a disialylated core type 2 hexasaccharide. In addition, N-linked site-specific information was obtained by MALDI-MS analysis using tryptic digestion after PNGase F release of the oligosaccharides.
authors.affiliation: Proteome Systems Ltd., 1/35-41 Waterloo Road, North Ryde, Sydney, NSW 1670, Australia. nwilson@proteomesystems.com
authors.forename: Nicole L
authors.initials: NL
authors.lastname: Wilson
authors.forename: Benjamin L
authors.initials: BL
authors.lastname: Schulz
authors.forename: Niclas G
authors.initials: NG
authors.lastname: Karlsson
authors.forename: Nicolle H
authors.initials: NH
authors.lastname: Packer
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005417:Flavobacterium; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D014357:Trypsin; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase