State‐of‐the‐art glycosaminoglycan characterization

Abstract

Glycosaminoglycans (GAGs) are heterogeneous acidic polysaccharides involved in a range of biological functions. They have a significant influence on the regulation of cellular processes and the development of various diseases and infections. To fully understand the functional roles that GAGs play in mammalian systems, including disease processes, it is essential to understand their structural features. Despite having a linear structure and a repetitive disaccharide backbone, their structural analysis is challenging and requires elaborate preparative and analytical techniques. In particular, the extent to which GAGs are sulfated, as well as variation in sulfate position across the entire oligosaccharide or on individual monosaccharides, represents a major obstacle. Here, we summarize the current state-of-the-art methodologies used for GAG sample preparation and analysis, discussing in detail liquid chromatograpy and mass spectrometry-based approaches, including advanced ion activation methods, ion mobility separations and infrared action spectroscopy of mass-selected species.

Glycosaminoglycans: What Remains To Be Deciphered?

“…Helium droplets pick up trapped ions, which are immediately cooled to 0.37 K. There is subsequent irradiation of the droplets with monochromatic, high-intensity IR radiation, for example, using a free-electron laser (FEL). Reproduced with permission from ref ( 71 ). Copyright 2021, John Wiley and Sons.…”

Predicting Structural Motifs of Glycosaminoglycans using Cryogenic Infrared Spectroscopy and Random Forest

“…5 The overall composition of a GAG chain can be assessed using lyase digestion and subsequent liquid chromatography of the resulting disaccharides. 6 Mass spectrometry-based experiments coupled with liquid chromatography, ion mobility, and different fragmentation techniques can further be used to identify characteristic oligosaccharide motifs, 4,7−9 although the analysis of the fragmentation data is a complex task and may not always lead to unambiguous assignments. 4 In the past few years, gas-phase vibrational spectroscopy of GAGs, and glycoconjugates in general, has emerged as a tool that provides complementary information to mass-spectrometry-based sequencing.…”

“…Generally, GAGs are divided into four major classes: heparin/heparan sulfate (Hp/HS), chondroitin sulfate/dermatan sulfate (CS/DS), keratan sulfate (KS), and hyaluronic acid (HA), with HS and CS exhibiting more diverse structures in comparison to KS and HA (Figure a). , Sequencing GAGs is of paramount importance to understand their physiological activity and interactions; however, it remains challenging due to their structural complexity and the limited availability of pure samples . The overall composition of a GAG chain can be assessed using lyase digestion and subsequent liquid chromatography of the resulting disaccharides . Mass spectrometry-based experiments coupled with liquid chromatography, ion mobility, and different fragmentation techniques can further be used to identify characteristic oligosaccharide motifs, ,− although the analysis of the fragmentation data is a complex task and may not always lead to unambiguous assignments …”

Extension of the SUGRES-1P Coarse-Grained Model of Polysaccharides to Heparin

“…The structure and dynamics of HP as well as its interactions with proteins have been investigated by various experimental techniques, including nuclear magnetic resonance (NMR), − X-ray diffraction, − surface plasmon resonance, − mass spectrometry, , and capillary electrophoresis. − However, experimental techniques face a range of challenges in the study of HP and other GAGs, mainly stemming from the periodicity, considerable length, and high molecular weight of native GAGs, − diversity of sequences and sulfation patterns, conformational flexibility, and tendency to cause oligomerization and precipitation of proteins …”