Abstract
Capillary electrophoresis is a powerful methodology for quantification and structural characterization of highly anionic polysaccharides. Separation of saccharides under conditions of electrophoretic flow, typically achieved under low pH (Ampofo et al., Anal Biochem 199: 249–255, 1991; Rhomberg et al., Proc Natl Acad Sci U S A 95: 4176–4181, 1998) is charge-based. Resolution of components is often superior to flow-based techniques, such as liquid chromatography. During the heparin contamination crisis, capillary electrophoresis was one of the key methodologies used to identify whether or not heparin lots were contaminated (Guerrini et al., Nat Biotechnol 26: 669–675, 2008; Ye et al., J Pharm Biomed Anal 85: 99–107, 2013; Volpi et al., Electrophoresis 33: 1531–1537, 2012).Here we describe a method for the isolation of sulfated heparin/heparan sulfate saccharides from urine, their digestion by deployment of heparinase enzymes (Ernst et al., Crit Rev Biochem Mol Biol 30: 387–444, 1995) resolution of species through use of orthogonal digestions, and analysis of the resulting disaccharides by capillary electrophoresis.
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References
Petitou M et al (1999) Synthesis of thrombin-inhibiting heparin mimetics without side effects. Nature 398:417–422
Zacharski LR, Ornstein DL (1998) Heparin and cancer. Thromb Haemost 80:10–23
Anderson VR, Perry CM (2006) Pentosan polysulfate: a review of its use in the relief of bladder pain or discomfort in interstitial cystitis. Drugs 66:821–835
Robinson LN et al (2012) Harnessing glycomics technologies: integrating structure with function for glycan characterization. Electrophoresis 33:797–814
Shukla D et al (1999) A novel role for 3-O-sulfated heparan sulfate in herpes simplex virus 1 entry. Cell 99:13–22
Tandon R et al (2020) Effective inhibition of SARS-CoV-2 entry by heparin and enoxaparin derivatives. bioRxiv
Nelson RM et al (1993) Heparin oligosaccharides bind L- and P-selectin and inhibit acute inflammation. Blood 82:3253–3258
Sundaram M et al (2003) Rational design of low-molecular weight heparins with improved in vivo activity. Proc Natl Acad Sci U S A 100:651–656
Schirm B, Benend H, Watzig H (2001) Improvements in pentosan polysulfate sodium quality assurance using fingerprint electropherograms. Electrophoresis 22:1150–1162
Yates EA et al (1996) 1H and 13C NMR spectral assignments of the major sequences of twelve systematically modified heparin derivatives. Carbohydr Res 294:15–27
Rhomberg AJ, Ernst S, Sasisekharan R, Biemann K (1998) Mass spectrometric and capillary electrophoretic investigation of the enzymatic degradation of heparin-like glycosaminoglycans. Proc Natl Acad Sci U S A 95:4176–4181
Kuberan B et al (2002) Analysis of heparan sulfate oligosaccharides with ion pair-reverse phase capillary high performance liquid chromatography-microelectrospray ionization time-of-flight mass spectrometry. J Am Chem Soc 124:8707–8718
Pepi LE, Sasiene ZJ, Mendis PM, Jackson GP, Amster IJ (2020) Structural characterization of sulfated glycosaminoglycans using charge transfer dissociation. J Am Soc Mass Spectrom 30(12):2692–2703
Beccati D et al (2017) An integrated approach using orthogonal analytical techniques to characterize heparan sulfate structure. Glycoconj J 34:107–117
Nemes P, Hoover WJ, Keire DA (2013) High-throughput differentiation of heparin from other glycosaminoglycans by pyrolysis mass spectrometry. Anal Chem 85:7405–7412
Kandrotas RJ (1992) Heparin pharmacokinetics and pharmacodynamics. Clin Pharmacokinet 22:359–374
Ampofo SA, Wang HM, Linhardt RJ (1991) Disaccharide compositional analysis of heparin and heparan sulfate using capillary zone electrophoresis. Anal Biochem 199:249–255
Khan SA, Mason RW, Kobayashi H, Yamaguchi S, Tomatsu S (2020) Advances in glycosaminoglycan detection. Mol Genet Metab 130:101–109
Aich U, Shriver Z, Tharakaraman K, Raman R, Sasisekharan R (2011) Competitive inhibition of heparinase by persulfonated glycosaminoglycans: a tool to detect heparin contamination. Anal Chem 83:7815–7822
Godavarti R et al (1996) Heparinase III from Flavobacterium heparinum: cloning and recombinant expression in Escherichia coli. Biochem Biophys Res Commun 225:751–758
Ernst S et al (1996) Expression in Escherichia coli, purification and characterization of heparinase I from Flavobacterium heparinum. Biochem J 315(Pt 2):589–597
Sasisekharan R, Bulmer M, Moremen KW, Cooney CL, Langer R (1993) Cloning and expression of heparinase I gene from Flavobacterium heparinum. Proc Natl Acad Sci U S A 90:3660–3664
Myette JR et al (2003) The heparin/heparan sulfate 2-O-sulfatase from Flavobacterium heparinum. Molecular cloning, recombinant expression, and biochemical characterization. J Biol Chem 278:12157–12166
Myette JR et al (2002) Molecular cloning of the heparin/heparan sulfate delta 4,5 unsaturated glycuronidase from Flavobacterium heparinum, its recombinant expression in Escherichia coli, and biochemical determination of its unique substrate specificity. Biochemistry 41:7424–7434
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This work was funded in part by National Institutes of Health (R37 GM057073-13 to RS).
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Shriver, Z., Sasisekharan, R. (2022). Capillary Electrophoretic Analysis of Isolated Sulfated Polysaccharides to Characterize Pharmaceutical Products. In: Balagurunathan, K., Nakato, H., Desai, U., Saijoh, Y. (eds) Glycosaminoglycans. Methods in Molecular Biology, vol 2303. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1398-6_27
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DOI: https://doi.org/10.1007/978-1-0716-1398-6_27
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