Small-scale enzymatic digestion of glycoproteins and proteoglycans for analysis of oligosaccharides by LC-MS and FACE gel electrophoresis.
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.
Estrella
Ruby P
RP
Graduate School of Biomedical Engineering, The University of New South Wales, Sydney, NSW, Australia. ruby.estrella@student.unsw.edu.au
Whitelock
John M
JM
Roubin
Rebecca H
RH
Packer
Nicolle H
NH
Karlsson
Niclas G
NG
D000818:Animals; D002240:Carbohydrate Sequence; D002853:Chromatography, Liquid / Q000379:methods; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004798:Enzymes / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000494:pharmacology; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008954:Models, Biological; D009844:Oligosaccharides / Q000302:isolation & purification* / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000502:physiology; D011509:Proteoglycans / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*
D004798:Enzymes; D005456:Fluorescent Dyes; D006023:Glycoproteins; D009844:Oligosaccharides; D011509:Proteoglycans