title: Top-down approach for the direct characterization of low molecular weight heparins using LC-FT-MS.
abstract: Low molecular heparins (LMWHs) are structurally complex, heterogeneous, polydisperse, and highly negatively charged mixtures of polysaccharides. The direct characterization of LMWH is a major challenge for currently available analytical technologies. Electrospray ionization (ESI) liquid chromatography-mass spectrometry (LC-MS) is a powerful tool for the characterization complex biological samples in the fields of proteomics, metabolomics, and glycomics. LC-MS has been applied to the analysis of heparin oligosaccharides, separated by size exclusion, reversed phase ion-pairing chromatography, and chip-based amide hydrophilic interaction chromatography (HILIC). However, there have been limited applications of ESI-LC-MS for the direct characterization of intact LMWHs (top-down analysis) due to their structural complexity, low ionization efficiency, and sulfate loss. Here we present a simple and reliable HILIC-Fourier transform (FT)-ESI-MS platform to characterize and compare two currently marketed LMWH products using the top-down approach requiring no special sample preparation steps. This HILIC system relies on cross-linked diol rather than amide chemistry, affording highly resolved chromatographic separations using a relatively high percentage of acetonitrile in the mobile phase, resulting in stable and high efficiency ionization. Bioinformatics software (GlycReSoft 1.0) was used to automatically assign structures within 5-ppm mass accuracy.
authors.affiliation: Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York 12180-3590, United States.
authors.forename: Lingyun
authors.initials: L
authors.lastname: Li
authors.forename: Fuming
authors.initials: F
authors.lastname: Zhang
authors.forename: Joseph
authors.initials: J
authors.lastname: Zaia
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Linhardt
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000032:analysis*; D002240:Carbohydrate Sequence; D002853:Chromatography, Liquid / Q000379:methods; D019295:Computational Biology / Q000379:methods; D017984:Enoxaparin / Q000032:analysis; D005583:Fourier Analysis; D006495:Heparin, Low-Molecular-Weight / Q000032:analysis*; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D012984:Software; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013552:Swine
chemical_list: D000925:Anticoagulants; D017984:Enoxaparin; D006495:Heparin, Low-Molecular-Weight