Mass spectrometric evidence for the mechanism of free-radical depolymerization of various types of glycosaminoglycans

Abstract Glycosaminoglycans (GAGs) are large, complex carbohydrate molecules that interact with a wide range of proteins involved in physiological and pathological processes. Several naturally derived GAGs have emerged as potentially useful therapeutics in clinical applications. Natural polysaccharides, however, generally have high molecular weights with a degree of polydispersity, making it difficult to investigate their structural properties. In this study, we establish a free-radical–mediated micro-reaction system and use hydrophilic interaction chromatography (HILIC)–Fourier transform mass spectrometry (FTMS) to profile the degraded products of various types of GAGs, heparin, chondroitin sulfate A, NS-heparosan, and oversulfated chondroitin sulfate (OSCS), to reveal the free-radical degradation mechanism of GAGs. The results show that the bulk fragments of GAGs generated by free-radical degradation can maintain their basic structural units and sulfate substituents. In addition, an abundance of oligomers modified with oxidation at their reducing ends or by dehydration also appeared. We discovered that these modifications were related in terms of the degree of sulfation and the α- or β-linkage of HexNY (Y = SO3− or Ac), and especially that the different linkage of the disaccharide unit is the main factor in modification. In addition, the method based on micro-free-radical reaction and HILIC-FTMS is both effective and sensitive, thus suggesting its broad practical value for the structural characterization and in the biological structure-function studies of GAGs.