Analysis of Glycosaminoglycans Using Mass Spectrometry

Abstract

The glycosaminoglycans (GAGs) are linear polysaccharides expressed on animal cell surfaces and in extracellular matrices. Their biosynthesis is under complex control and confers a domain structure that is essential to their ability to bind to protein partners. Key to understanding the functions of GAGs are methods to determine accurately and rapidly patterns of sulfation, acetylation and uronic acid epimerization that correlate with protein binding or other biological activities. Mass spectrometry (MS) is particularly suitable for the analysis of GAGs for biomedical purposes. Using modern ionization techniques it is possible to accurately determine molecular weights of GAG oligosaccharides and their distributions within a mixture. Methods for direct interfacing with liquid chromatography have been developed to permit online mass spectrometric analysis of GAGs. New tandem mass spectrometric methods for fine structure determination of GAGs are emerging. This review summarizes MS-based approaches for analysis of GAGs, including tissue extraction and chromatographic methods compatible with LC/MS and tandem MS.

Keywords: Mass spectrometry; chondroitin sulfate; dermatan sulfate; glycosaminoglycan; heparan sulfate; heparin; hyaluronan; keratan sulfate; proteoglycan.

Figure 1

Structures of the glycosaminoglycan classes. The nascent polysaccharide repeats are given in parenthesis in the figure. Hyaluronan is expressed as an unmodified polysaccharide. The mature HS chains have some uronic acid residues epimerized to IdoA. Sulfation of HS may occur at the 2O-position of uronic acids and at the N-, 3O-, and 6O-positions of GlcN. CS chains may undergo epimerization to create IdoA residues. Sulfation may occur at the 2O-position of uronic acids, and the 4O- and 6O-positions of GalNAc residues. KS chains are sulfated at many 6O-positions of GlcNAc and 6O-positions of Gal residues.

Figure 2

Structures for Arixtra (A) and disulfated CCR2 21–30 peptide (B)

Figure 3

Mass spectra of C-C motif chemokine 7 (CCL7) and Arixtra with the addition of competing ligand, disulfated CCR2 21–30. The upper spectrum (A) represents a sample containing 10 μM CCL7, 10 μM Arixtra, and 1 μM disulfated CCR2. The lower spectrum (B) represents a sample containing 10 μM CCL7, 10 μM Arixtra, and 10 μM disulfated CCR2. Structures for Arixtra and disulfated CCR2 21–30 peptide are defined in Figure 2. © 2010, Elsevier, Inc. Used with permission.