Optimized extraction of glycosaminoglycans from normal and osteoarthritic cartilage for glycomics profiling

Abstract

Articular cartilage is a highly specialized smooth connective tissue whose proper functioning depends on the maintenance of an extracellular matrix consisting of an integrated assembly of collagens, glycoproteins, proteoglycans (PG), and glycosaminoglycans. Isomeric chondroitin sulfate glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work introduces a novel glycosaminoglycan extraction method for the quantification of mixtures of chondroitin sulfate oligosaccharides from intact cartilage tissue for mass spectral analysis. Glycosaminoglycans were extracted from intact cartilage samples using a combination of ethanol precipitation and enzymatic release followed by reversed-phase and strong anion exchange solid-phase extraction steps. Extracted chondroitin sulfate glycosaminoglycans were partially depolymerized using chondroitinases, labeled with 2-anthranilic acid-d(4) (2-AA) and subjected to size exclusion chromatography with online electrospray ionization mass spectrometric detection in the negative ion mode. The method presented herein enabled simultaneous determination of sulfate position and uronic acid epimerization in juvenile bovine and adult human cartilage samples. The method was applied to a series of 13 adult human cartilage explants. Standard deviation of the mean for the measurements was 1.6 on average. Coefficients of variation were approximately 4% for all compositions of 40% or greater. These results show that the new method has sufficient accuracy to allow determination of topographical distribution of glycoforms in connective tissue.

Organ-specific Heparan Sulfate Structural Phenotypes

“…SEC LC/MS Analysis of Native HS Disaccharides-SEC has advantages for disaccharide analysis over adsorption chromatographic modes (32,35,38). The disaccharides are separated based on hydrodynamic volume, roughly proportional to the number of sulfate groups present.…”

“…Our laboratory has developed an LC/MS/MS platform to analyze oligosaccharides derived from chondroitin sulfate from connective tissue (35,36). Analysis of GAGs from animal organs, however, is more challenging due to higher fat content and variability in fibrous structure.…”

Evolutionary Differences in Glycosaminoglycan Fine Structure Detected by Quantitative Glycan Reductive Isotope Labeling

“…To circumvent these problems, LC/MS procedures have been developed that utilize linear equations to correlate the concentration of underivatized disaccharides with the detection of molecular standards (10,(17)(18)(19)(20). Another approach is to use mass-tagging techniques based on differentially isotopically labeled tags introduced into oligosaccharides derived from intact chains (16,(21)(22)(23)(24). Here we describe a mass-tagging technique that allows quantitative analysis of disaccharide composition and ratiometric comparisons between samples.…”

The extracellular matrix: an underexplored but important proteome

“…Mass spectrometry has played a major role in the analysis of modifications, including tyrosine sulfation (osteoadherin, fibromodulin and lumican), N-linked glycan structures (cartilage oligomeric matrix protein) and disease-related changes in GAG sulfation (aggrecan) [9][10][11][12]. In collagens, age-and tissue-specific patterns of proline/lysine hydroxylation and O-linked glycosylation critically influence fibril crosslinking and tensile strength [13].…”