Small-scale enzymatic digestion of glycoproteins and proteoglycans for analysis of oligosaccharides by LC-MS and FACE gel electrophoresis

Ruby P. Estrella*, John M. Whitelock, Rebecca H. Roubin, Nicolle H. Packer, Niclas G. Karlsson

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

8 Citations (Scopus)

Abstract

Structural characterization of oligosaccharides from proteoglycans and other glycoproteins is greatly enhanced through the use of mass spectrometry and gel electrophoresis. Sample preparation for these sensitive techniques often requires enzymatic treatments to produce oligosaccharide sequences for subsequent analysis. This chapter describes several small-scale methods for in-gel, on-blot, and in-solution enzymatic digestions in preparation for graphitized carbon liquid chromatography-mass spectrometry (LC-MS) analysis, with specific applications indicated for glycosaminoglycans (GAGs) and N-linked oligosaccharides. In addition, accompanying procedures for oligosaccharide reduction by sodium borohydride, sample desalting via carbon microcolumn, desialylation by sialidase enzyme treatment, and small-scale oligosaccharide species fractionation are included. Fluorophore-assisted carbohydrate electrophoresis (FACE) is another useful method to isolate derivatized oligosaccharides. Overall, the modularity of these techniques provides ease and flexibility for use in conjunction with mass spectrometric and electrophoretic tools for glycomic research studies.

Original languageEnglish
Title of host publicationGlycomics
Subtitle of host publicationMethods and protocols
EditorsNicolle H. Packer, Niclas G. Karlsson
Place of PublicationTotowa, N.J
Pages171-192
Number of pages22
Volume534
ISBN (Electronic)9781597450225
DOIs
Publication statusPublished - 2009
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume534
ISSN (Print)10643745

Keywords

  • Fluorophore-assisted carbohydrate electrophoresis
  • Glycosaminoglycans
  • Graphitized carbon LC-MS
  • In-gel enzyme digestion
  • N-linked oligosaccharides
  • On-blot enzyme digestion

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