Glycosaminoglycanomics of Cultured Cells Using a Rapid and Sensitive LC-MS/MS Approach

Glycosaminoglycans (GAGs), a family of polysaccharides widely distributed in eukaryotic cells, are responsible for a wide array of biological functions. Quantitative disaccharide compositional analysis is one of the primary ways to characterize the GAG structure. This structural analysis is typically time-consuming (1–2 weeks) and labor intensive, requiring GAG recovery and multistep purification, prior to the enzymatic/chemical digestion of GAGs, and finally their analysis. Moreover, 10⁵–10⁷ cells are usually required for compositional analysis. We report a sensitive, rapid, and quantitative analysis of GAGs present in a small number of cells. Commonly studied cell lines were selected based on phenotypic properties related to the biological functions of GAGs. These cells were lysed using a commercial surfactant reagent, sonicated, and digested with polysaccharide lyases. The resulting disaccharides were recovered by centrifugal filtration, labeled with 2-aminoacridone, and analyzed by liquid chromatography (LC)-mass spectrometry (MS). Using a highly sensitive MS method, multiple reaction monitoring (MRM), the limit of detection for each disaccharide was reduced to 0.5–1.0 pg, as compared with 1.0–5.0 ng obtained using standard LC-MS analysis. Sample preparation time was reduced to 1–2 days, and the cell number required was reduced to 5000 cells for complete GAG characterization to as few as 500 cells for the characterization of the major GAG disaccharide components. Our survey of the glycosaminoglycanomes of the 20 selected cell lines reveals major differences in their GAG amounts and compositions. Structure–function relationships are explored using these data, suggesting the utility of this method in cellular glycobiology

Bottom-Up Low Molecular Weight Heparin Analysis Using Liquid Chromatography-Fourier Transform Mass Spectrometry for Extensive Characterization

Low molecular weight heparins (LMWHs) are heterogeneous, polydisperse, and highly negatively charged mixtures of glycosaminoglycan chains prescribed as anticoagulants. The detailed characterization of LMWH is important for the drug quality assurance and for new drug research and development. In this study, online hydrophilic interaction chromatography (HILIC) Fourier transform mass spectrometry (FTMS) was applied to analyze the oligosaccharide fragments of LMWHs generated by heparin lyase II digestion. More than 40 oligosaccharide fragments of LMWH were quantified and used to compare LMWHs prepared by three different manufacturers. The quantified fragment structures included unsaturated disaccharides/oligosaccharides arising from the prominent repeating units of these LMWHs, 3-<i>O</i>-sulfo containing tetrasaccharides arising from their antithrombin III binding sites, 1,6-anhydro ring-containing oligosaccharides formed during their manufacture, saturated uronic acid oligosaccharides coming from some chain nonreducing ends, and oxidized linkage region oligosaccharides coming from some chain reducing ends. This bottom-up approach provides rich detailed structural analysis and quantitative information with high accuracy and reproducibility. When combined with the top-down approach, HILIC LC-FTMS based analysis should be suitable for the advanced quality control and quality assurance in LMWH production

Method to Detect Contaminants in Heparin Using Radical Depolymerization and Liquid Chromatography–Mass Spectrometry

Heparin is a critically important anticoagulant drug that was contaminated with a persulfonated polysaccharide in 2008, resulting in a number of severe adverse reactions, some leading to death. Controversy remains as to the precise composition of the 2008 contaminant, and new information suggests that heparin may now be subject to adulteration with a new, difficult to detect, contaminant, <i>N</i>-sulfo oversulfated chondroitin sulfate. This study synthesizes this new potential contaminant and describes the use of radical depolymerization followed by liquid chromatography–mass spectrometry to detect N-sulfo oversulfated chondroitin sulfate and to confirm the structure of the 2008 contaminant as oversulfated chondroitin sulfate and not oversulfated heparan sulfate