Coupling Liquid Chromatography and Tandem Mass Spectrometry to Electrophoresis for In-Depth Analysis of Glycosaminoglycan Drugs: Heparin and the Multicomponent Sulodexide

Abstract

Glycosaminoglycans (GAGs) contribute to the treatment of many human diseases, especially in the field of thrombosis, because of their anticoagulant activity. GAGs interrupt the coagulation process by interacting with multiple coagulation factors through defined sequences within their linear and negatively charged chains, which are not fully elucidated. Numerous methods have been developed to characterize the structure of pharmaceutical GAGs, including intravenously or subcutaneously administered heparin and orally administered sulodexide. However, most currently available methods only focus on the oligosaccharide portion or analyze the whole mixture because longer-chain polysaccharides are extremely difficult to resolve by chromatographic separation. We have established two novel electrophoresis–mass spectrometry methods to provide a panoramic view of the structures of pharmaceutical GAGs. In the first method, an in-gel digestion procedure was developed to recover GAGs from the polyacrylamide gels, while in the second method, a strong anion exchange ultrafiltration procedure was developed to extract multiple GAG species from the agarose gels. Both procedures are compatible with liquid chromatography–tandem mass spectrometry, and structural information, such as disaccharide composition and chain length, can be revealed for each GAG fraction. The applications of these two methods on analysis of two different GAG drugs, heparin and sulodexide, were demonstrated. The current study offers the first robust tool to directly elucidate the structure of larger GAG chains with more biological importance rather than obtaining a vague picture of all chains as a mixture, which is fundamental for better understanding the structure–activity relationship and quality control of the GAG drugs.

Imaging of Escherichia coli K5 and glycosaminoglycan precursors via targeted metabolic labeling of capsular polysaccharides in bacteria

“…S1), followed by the introduction of PmHS2 under the control of an inducible promoter. Disaccharide analysis based on the degradation of polysaccharides by heparosan endo-β-eliminase HepIII ( 26 ) demonstrated that KfiA Δ strains lost K5 capsule-synthesis activity, but expression of PmHS2 could rescue this disability (fig. S2).…”

“…To verify that azido and alkyne incorporation was limited to K5 capsular polysaccharide, we subjected the GlcNAz- and GlcNAl-supplemented K5ASSH cells to digestion with heparosan endo-β-eliminase HepIII ( 26 ), which specifically targets the α-1,4 glycosidic bonds between GlcNAc (or its analogs) and GlcA residues. We observed an overall downshift, almost disappearance, of the azide-dependent or alkyne-dependent fluorescence, consistent with degradation of the heparosan (Fig.…”

“…1C). To further verify the nonnatural sugar labeling of the capsule, K5 capsular polysaccharide was isolated from the GlcNAz-supplemented strain K5ASSH and incubated with HepIII; the resulting disaccharides were examined by high-performance liquid chromatography–mass spectrometry (HPLC-MS) ( 26 ) (fig. S6).…”

“…After the reaction was terminated, the supernatant was taken, and salt was removed by 3-kDa ultrafiltration, yielding GAG polysaccharides produced by bacteria. Then, disaccharides were obtained by enzymolysis with the corresponding GAG endo-eliminase ( 26 , 35 ). The heparosan was suspended in 200 μl of tristeamed water (0.1 mg/ml) containing 10 mM CaCl 2 , 50 mM tris-HCl (pH 7.0), and heparin endo-eliminase HepIII (0.2 mg/ml).…”

A Species-Specific Strategy for the Identification of Hemocoagulase Agkistrodon halys pallas Based on LC-MS/MS-MRM

“…Mass spectrometry plays a key role in the structural characterization of glycans, proteins, glycan–protein interactions, and protein–protein interactions ( Britt et al, 2022 ; Grabarics et al, 2022 ; Sheng et al, 2021 ; Zhang and Chi, 2021 ). Shotgun proteomics based on liquid chromatography−tandem mass spectrometry (LC−MS/MS) and database search are widely used for the qualitative and quantitative analysis of proteins in complex biological samples ( Huang and Wang, 2021 ; Marques et al, 2021 ).…”