Multidose Inline Size-Exclusion Chromatography–Flash Oxidation for Structural Analysis of Dynamic Protein–Ligand Interactions of Antithrombin III and Unfractionated Heparin

Hydroxyl radical protein footprinting (HRPF) is a well-established technique used to analyze the binding sites and conformational alterations of proteins. Inline LC-flash oxidation can efficiently capture and characterize structural changes between dynamic protein-ligand complexes as they elute from the LC column. The current work focuses on the development of multidose inline SEC-FOX (size-exclusion chromatography-flash oxidation) for the quantitative topographical analysis of the dynamic protein-ligand complex of human antithrombin III (ATIII) and unfractionated heparin (UFH). ATIII alone and the ATIII-UFH complex were separated by an inline SEC system and flash oxidized at multiple radical doses, followed by proteolytic digestion and LC-MS/MS analysis. A total of 21 oxidized peptides, including three glycopeptides with two major glycoforms each, were reproducibly detected. Six peptides including one glycopeptide (48-53, 58-70, 189-197 A2G2S2 glycoform, 108-114, 333-348, and 360-370) were significantly protected from oxidation upon UFH binding, and one peptide (242-257) showed a significant increase in oxidation upon UFH binding. Residue-level analysis showed that three amino acids (M49, E113, and F329) were significantly protected from modification in the ATIII-UFH complex compared to ATIII alone. Two of these three amino acids are proximal to the known pentasaccharide binding site, while the third is distal and suggests a potential extended interaction of the full-length UFH with ATIII, which was well supported by computational modeling data. We interpret these findings in the context of previously published biophysical data and present a computational model of UFH binding consistent with our findings.