Sequential analysis of N- and O-linked glycosylation of 2D-PAGE separated glycoproteins

Nicole L. Wilson*, Benjamin L. Schulz, Niclas G. Karlsson, Nicolle H. Packer

*Corresponding author for this work

Research output: Contribution to journalArticle

117 Citations (Scopus)

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 β-elimination and analysis using LCESI-MS. Oligosaccharides from the major plasma glycoproteins with a p/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.

Original languageEnglish
Pages (from-to)521-529
Number of pages9
JournalJournal of Proteome Research
Volume1
Issue number6
DOIs
Publication statusPublished - Nov 2002
Externally publishedYes

Keywords

  • β-elimination
  • Glycoproteins
  • Plasma
  • PNGase F
  • Sialylation

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