Disaccharide analysis of glycosaminoglycan mixtures by ultra-high-performance liquid chromatography–mass spectrometry

Abstract

Glycosaminoglycans are a family of polysaccharides widely distributed in all eukaryotic cells. These polyanionic, linear chain polysaccharides are composed of repeating disaccharide units that are often differentially substituted with sulfo groups. The diversity of glycosaminoglycan structures in cells, tissues and among different organisms reflect their functional an evolutionary importance. Glycosaminoglycan composition and structure also changes in development, aging and in disease progression, making their accurate and reliable analysis a critical, albeit, challenging endeavor. Quantitative disaccharide compositional analysis is one of the primary ways to characterize glycosaminoglycan composition and structure and has a direct relationship with glycosaminoglycan biological functions. In this study, glycosaminoglycan disaccharides, prepared from heparan sulfate/heparin, chondroitin sulfate/dermatan sulfate and neutral hyaluronic acid using multiple polysaccharide lyases, were fluorescently labeled with 2-aminoacridone, fractionated into 17 well-resolved components by reverse-phase ultra-performance liquid chromatography, and analyzed by electrospray ionization mass spectrometry. This analysis was successfully applied to cell, tissue, and biological fluid samples for the picomole level detection of glycosaminoglycan composition and structure.

The Circulating Glycosaminoglycan Signature of Respiratory Failure in Critically Ill Adults

“…We have developed a highly specific analytical methodology for the analysis of glycosaminoglycan-derived disaccharides that is based on specific polysaccharide lyase digestion, AMAC-labeling of the disaccharides at their hemiacetal reducing ends, and LC-MS detection (16). This results in improved signal to noise over previous methods (28) and affords a detection limit as low as 100 pg of disaccharide in a plasma sample.…”

“…, and HA disaccharides were performed on an Agilent 1200 LC/MSD instrument (Agilent Technologies, Inc. Wilmington, DE) equipped with a 6300 iontrap and a binary pump, as described previously (16). Briefly, the column used was a Poroshell 120 C18 column (3.0 ϫ 150 mm, 2.7 m, Agilent) at 45°C.…”

“…The electrospray interface was set in negative ionization mode with a skimmer potential of Ϫ40.0 V, a capillary exit of Ϫ40.0 V, and a source temperature of 350°C, to obtain the maximum abundance of the ions in a full-scan spectrum (150 -1200 Da). Nitrogen (8 liters/ min, 40 psi) was used as a drying and nebulizing gas (16).…”

“…enzymatic cleavage versus nonspecific destruction (2, 15)) could impart characteristic glycosaminoglycan fragment patterns. Novel mass spectrometry techniques offer both precise quantification and characterization of circulating glycosaminoglycans (16), allowing for the detection of signatures of ESL degradation that may have relevance as biomarkers of critical illness.…”

Fibroblast Growth Factor-based Signaling through Synthetic Heparan Sulfate Blocks Copolymers Studied Using High Cell Density Three-dimensional Cell Printing

“…The disaccharide compositions of block co-polymers were analyzed by high performance liquid chromatography-mass spectrometry (57). Briefly, the block copolymers (5 g) were incubated with a mixture of heparin/HS lyase I, II, and III (10 milliunits each) at 37°C for 10 h. The digested disaccharides were recovered by passing through a 30,000-MWCO Amicon Ultra spin unit at 12,000 ϫ g for 10 min.…”