title: Characterization of conformers and dimers of antithrombin by capillary electrophoresis-quadrupole-time-of-flight mass spectrometry.
abstract: Antithrombin (AT) is a plasma glycoprotein which possesses anticoagulant and anti-inflammatory properties. AT exhibits various forms, among which are native, latent and heterodimeric ones. We studied the potential of capillary electrophoresis-mass spectrometry (CE-MS) using a sheath liquid interface, electrospray ionization (ESI), and a quadrupole-time-of-flight (Q-TOF) mass spectrometer to separate and quantify the different AT forms. For CE separation, a neutral polyvinyl alcohol (PVA) coated capillary was employed. The protein conformation was preserved by using a background electrolyte (BGE) at physiological pH. A sheath liquid of isopropanol-water 50:50 (v/v) with 14 mM ammonium acetate delivered at a flow rate of 120 μL h-1resulted in optimal signal intensities. Each AT form exhibited a specific mass spectrum, allowing unambiguous distinction. Several co-injection experiments proved that latent AT had a higher electrophoretic mobility (μep) than native AT, and that these conformers could associate to form a heterodimer during the CE analysis. The developed CE-MS method enabled the detection and quantitation of latent and heterodimeric forms in a commercial AT preparation stored at room temperature for three weeks.
authors.affiliation: Institut Galien Paris Sud, UMR8612, Protein and Nanotechnology in Analytical Science (PNAS), CNRS, Univ. Paris-Sud, Université Paris-Saclay, 5 rue Jean-Baptiste Clément, 92290, Châtenay-Malabry, France.
authors.forename: Anne-Lise
authors.initials: AL
authors.lastname: Marie
authors.affiliation: Division of BioAnalytical Chemistry, AIMMS Research Group Biomolecular Analysis, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands.
authors.forename: Elena
authors.initials: E
authors.lastname: Dominguez-Vega
authors.affiliation: Université Paris Sud, UMR-S1176, 94276, Le Kremlin-Bicêtre, France; INSERM, U1176, 94276, Le Kremlin-Bicêtre, France.
authors.forename: François
authors.initials: F
authors.lastname: Saller
authors.affiliation: LFB, 91940, Les Ulis, France.
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Plantier
authors.affiliation: ECDYSIS Pharma, 59120, Loos, France.
authors.forename: Rémi
authors.initials: R
authors.lastname: Urbain
authors.affiliation: Université Paris Sud, UMR-S1176, 94276, Le Kremlin-Bicêtre, France; INSERM, U1176, 94276, Le Kremlin-Bicêtre, France; AP-HP, Hôpital Necker, Service d'Hématologie Biologique, 75015, Paris, France.
authors.forename: Delphine
authors.initials: D
authors.lastname: Borgel
authors.affiliation: Institut Galien Paris Sud, UMR8612, Protein and Nanotechnology in Analytical Science (PNAS), CNRS, Univ. Paris-Sud, Université Paris-Saclay, 5 rue Jean-Baptiste Clément, 92290, Châtenay-Malabry, France.
authors.forename: N Thuy
authors.initials: NT
authors.lastname: Tran
authors.affiliation: Division of BioAnalytical Chemistry, AIMMS Research Group Biomolecular Analysis, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands.
authors.forename: Govert W
authors.initials: GW
authors.lastname: Somsen
authors.affiliation: Institut Galien Paris Sud, UMR8612, Protein and Nanotechnology in Analytical Science (PNAS), CNRS, Univ. Paris-Sud, Université Paris-Saclay, 5 rue Jean-Baptiste Clément, 92290, Châtenay-Malabry, France. Electronic address: myriam.taverna@u-psud.fr.
authors.forename: Myriam
authors.initials: M
authors.lastname: Taverna
mesh_terms: D058833:Antithrombin Proteins / Q000737:chemistry* / Q000302:isolation & purification; D019075:Electrophoresis, Capillary / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D013696:Temperature
chemical_list: D058833:Antithrombin Proteins
keywords: Antithrombin; Capillary electrophoresis-mass spectrometry; Conformers; Heterodimer; Latent form