title: Heparin Isomeric Oligosaccharide Separation Using Volatile Salt Strong Anion Exchange Chromatography.
abstract: The complexity of heparin and heparan sulfate saccharides makes their purification, including many isomeric structures, very challenging and is a bottleneck for structure-activity studies. High-resolution separations have been achieved by strong anion exchange (SAX) chromatography on Propac PA1 and cetyltrimethylammonium (CTA)-C18silica columns; however, these entail subsequent desalting methodologies and consequent sample losses and are incompatible with orthogonal chromatography methodologies and, in particular, mass spectrometry. Here, we present the CTA-SAX purification of heparin oligosaccharides using volatile salt (VS) buffer. In VSCTA-SAX, the use of ammonium bicarbonate buffer for elution improves resolution through both weaker dissociation and conformational coordination of the ammonium across the sulfate groups. Using ion mobility mass spectrometry, we demonstrate that isomeric structures have different structural conformations, which makes chromatographic separation achievable. Resolution of such structures is improved compared to standard SAX methods, and in addition, VSCTA-SAX provides an orthogonal method to isolate saccharides with higher purity. Because ammonium bicarbonate is used, the samples can be evaporated rather than desalted, preventing substantial sample loss and allowing more effective subsequent analysis by electrospray mass spectrometry. We conclude that VSCTA-SAX is a powerful new tool that helps address the difficult challenge of heparin/heparan sulfate saccharide separation and will enhance structure-activity studies.
authors: Centre for Glycobiology, Department of Biochemistry, Institute of Integrative Biology, University of Liverpool , Crown Street, Liverpool L69 7ZB, United Kingdom.|Departments of Molecular and Cellular Biology and Chemistry, University of California , 1 Shields Drive, Davis, California 95616, United States.
authors: Rebecca L
authors: RL
authors: Miller
authors: Centre for Glycobiology, Department of Biochemistry, Institute of Integrative Biology, University of Liverpool , Crown Street, Liverpool L69 7ZB, United Kingdom.
authors: Scott E
authors: SE
authors: Guimond
authors: Centre for Glycobiology, Department of Biochemistry, Institute of Integrative Biology, University of Liverpool , Crown Street, Liverpool L69 7ZB, United Kingdom.
authors: Maitreyi
authors: M
authors: Shivkumar
authors: Centre for Glycobiology, Department of Biochemistry, Institute of Integrative Biology, University of Liverpool , Crown Street, Liverpool L69 7ZB, United Kingdom.
authors: Jemma
authors: J
authors: Blocksidge
authors: Centre for Glycobiology, Department of Biochemistry, Institute of Integrative Biology, University of Liverpool , Crown Street, Liverpool L69 7ZB, United Kingdom.
authors: James A
authors: JA
authors: Austin
authors: Departments of Molecular and Cellular Biology and Chemistry, University of California , 1 Shields Drive, Davis, California 95616, United States.
authors: Julie A
authors: JA
authors: Leary
authors: Centre for Glycobiology, Department of Biochemistry, Institute of Integrative Biology, University of Liverpool , Crown Street, Liverpool L69 7ZB, United Kingdom.
authors: Jeremy E
authors: JE
authors: Turnbull
mesh_terms: D002593:Cetrimonium Compounds / Q000737:chemistry; D002852:Chromatography, Ion Exchange; D006493:Heparin / Q000737:chemistry*; D000075663:Ion Mobility Spectrometry; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification*; D012492:Salts / Q000737:chemistry; D013237:Stereoisomerism; D055549:Volatile Organic Compounds / Q000737:chemistry*
chemical_list: D002593:Cetrimonium Compounds; D009844:Oligosaccharides; D012492:Salts; D055549:Volatile Organic Compounds; D006493:Heparin