title: Interactions of Intact Unfractionated Heparin with Its Client Proteins Can Be Probed Directly Using Native Electrospray Ionization Mass Spectrometry.
abstract: Heparin and related members of the glycosaminoglycan (GAG) family are highly polyanionic linear saccharides that play important roles in a variety of physiological processes ranging from blood coagulation to embryo- and oncogenesis, tissue regeneration, and immune response regulation. These diverse functions are executed via a variety of mechanisms, including protein sequestration, activation, and facilitation of their interactions with cell-surface receptors, but deciphering the specific molecular mechanisms is frequently impossible due to the extremely high degree of GAG heterogeneity. As a result, the vast majority of studies of heparin (or related GAGs) interactions with its client proteins use synthetically produced heparin mimetics with defined structure or short heparin fragments. In this work we use native electrospray ionization mass spectrometry (ESI MS) in combination with limited charge reduction in the gas phase to obtain meaningful information on noncovalent complexes formed by intact unfractionated heparin and antithrombin-III, interaction which is central to preventing blood clotting. Complexes of different stoichiometries are observed ranging from 1:1 to 1:3 (heparin/protein ratio). In addition to binding stoichiometry, the measurements allow the range of heparin chain lengths to be obtained for each complex and the contribution of each complex to the total ionic signal to be calculated. Incorporation of ion mobility measurements in the experimental workflow allows the total analysis time to be shortened very significantly and the charge state assignment for the charge-reduced species to be verified. The possibility to study interactions of intact unfractionated heparin with a client protein carried out directly by native ESI MS without the need to use relatively homogeneous surrogates demonstrated in this work opens up a host of new exciting opportunities and goes a long way toward ameliorating the persistent but outdated view of the intractability of such interactions.
authors.affiliation: Department of Chemistry, University of Massachusetts-Amherst , Amherst, Massachusetts 01003, United States.
authors.forename: Yunlong
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Department of Chemistry, University of Massachusetts-Amherst , Amherst, Massachusetts 01003, United States.
authors.forename: Rinat R
authors.initials: RR
authors.lastname: Abzalimov
authors.affiliation: Department of Chemistry, University of Massachusetts-Amherst , Amherst, Massachusetts 01003, United States.
authors.forename: Igor A
authors.initials: IA
authors.lastname: Kaltashov
mesh_terms: D000990:Antithrombin III / Q000737:chemistry*; D006025:Glycosaminoglycans / Q000737:chemistry*; D006493:Heparin / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D006025:Glycosaminoglycans; D000990:Antithrombin III; D006493:Heparin