Sequential Analysis of N- and O-Linked Glycosylation of 2D-PAGE Separated Glycoproteins

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...

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Bibliographic Details
Published in:Journal of proteome research 2002-11, Vol.1 (6), p.521-529
Main Authors: Wilson, Nicole L, Schulz, Benjamin L, Karlsson, Niclas G, Packer, Nicolle H
Format: Article
Language:English
Subjects:
Amidohydrolases - metabolism
Electrophoresis, Gel, Two-Dimensional
Electrophoresis, Polyacrylamide Gel
Flavobacterium
Glycoproteins - analysis
Glycoproteins - chemistry
Glycoproteins - metabolism
Glycosylation
Humans
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Trypsin - metabolism
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Summary: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 β-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, α2-HS-glycoprotein, serotransferrin, α1-antitrypsin, and α1-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. Keywords: sialylation • glycoproteins • β-elimination • PNGase F • plasma
ISSN:1535-3893
1535-3907
DOI:10.1021/pr025538d