Speaker: Igor A. Kaltashov, Professor, Department of Chemistry. University of Massachusetts Amherst
Title: New mass spectrometry-based approaches to characterization of highly heterogeneous macromolecules: focus on heparin
Abstract: Understanding molecular mechanisms governing interactions of glycosaminoglycans (such as heparin) with proteins remains challenging due to their enormous structural heterogeneity. Commonly accepted approaches seek to reduce this structural complexity by searching for “minimal protein-binding epitopes” within the limited subsets of short heparin oligomers produced either enzymatically or synthetically. An alternative approach presented in this work seeks to preserve the chemical diversity displayed by heparin by allowing the intact polysaccharide chains to interact with the client protein. Native mass spectrometry (MS) is used in combination with ion chemistry in the gas phase to characterize intact protein/heparin complexes, providing information on their composition and binding stoichiometry. More detailed structural information is obtained by carrying out enzymatic foot-printing in solution by lysing the protein-bound heparin chains followed by the product analysis using size exclusion chromatography with on-line MS detection. When applied to a paradigmatic heparin/antithrombin system, the new method generates a series of oligomers with surprisingly distinct sulfation levels, highlighting the important role of the electrostatics outside of the binding core (where the protein/heparin association is guided by structural complementarity). In addition to being an auxiliary force, electrostatic interactions may rescue protein/heparin association when the structurally complementary binders are unavailable. Applications of MS-based approaches to other therapeutically relevant protein/heparin systems are also considered.