Strategies for analysis and structure characterization of glycans/proteoglycans by capillary electrophoresis. Their diagnostic and biopharmaceutical importance

Abstract

Proteoglycans are key biological macromolecules that, via their glycan constituents, participate and regulate several cellular events and physiopathological processes. Refined structures of their highly anionic glycan chains, involving sulphation pattern and uronic acid distribution through the polymeric chain, determine the interactions of proteoglycans with matrix effector molecules and are responsible for numerous effects. Analysis and structural characterization of glycans are, therefore, essential in understanding the biological functions of proteoglycans. Capillary electrophoresis with its high resolving power and sensitivity may successfully be used for the fine chemical characterization of components present in low amounts and to overcome limitations due to low amount/volume of biologic samples available. This technique is also friendly to the user and to the environment, since a very small amount of solvents (a few microL) is required. In this review the strategies used to analyse and characterize the structure of glycan chains of proteoglycans are summarized. They involve capillary electrophoretic analysis of depolymerized acidic glycan chains using specific enzymes and analysis of intact chains. The importance of this type of analysis in biologic samples and tissues and its possible diagnostic and biopharmaceutical use are also discussed.

High‐performance CE of Escherichia coli K4 cell surface polysaccharides

“…To reach this goal, first of all, purified K4 and defructosylated CPS samples were analysed in our conditions at different concentrations. Differently from what has been reported in the literature, the two species showed a maximum of absorbance at 190 nm; this result was not surprising considering that this low UV wavelength has already been reported as the most suitable one for the detection of intact GAG chains, including the non‐sulphated hyaluronic acid 33, 34. Besides, as we noted, the 190 nm detection, compared with the 200 nm, allowed to quantify the two polysaccharides without underestimating their concentrations and resulting in a higher sensitivity of the method.…”

A modified protocol for isolation and purity evaluation of a staphylococcal acidic polysaccharide by chromatography and capillary electrophoresis

“…CS and HA are negatively charged molecules (one uronic acid per repeating disaccharide unit) of charge density depending on the presence of O‐sulfated groups. At a low pH of the operating buffer utilized, a reversed polarity was selected for the electrophoretic separation, as previously described (Karamanos and Hjerpe, 1999). Under the conditions used, analysis of CS by CE at 200 nm showed the presence of one peak migrating at 6 min, representing a homogeneously charged population.…”

Glycosylation States on Intact Proteins Determined by NMR Spectroscopy

“…To improve our understanding of structure/function and to ensure proper glycosylation of protein therapeutics, the glycans must be fully characterized. Typically, mass spectrometry (MS)-based methods are combined with other methods, such as glycan enrichment, affinity separation, enzymatic digestion, liquid chromatography (LC) and/or gas chromatography (GC), to determine protein glycosylation [ 24 , 25 , 26 ]. However, the stereochemistry of a glycan, including the type of glycosidic linkage, are challenging to determine by MS, because it is difficult to distinguish between isobaric species like glucose (Glc), galactose (Gal), and mannose (Man).…”