Exploration of PF4 Binding and Conformational Dynamics via Hydrogen-Deuterium Exchange Mass Spectrometry

Presenter

Daniel Heron

Campus

UMass Amherst

Sponsor

Igor Kaltashov, Department of Chemistry, UMass Amherst

Schedule

Session 4, 2:30 PM - 3:15 PM [Schedule by Time][Poster Grid for Time/Location]

Location

Poster Board A59, Campus Center Auditorium, Row 3 (A41-A60) [Poster Location Map]

Abstract

Heparin induced thrombocytopenia (HIT) is a potentially fatal complication of sustained heparin infusion – a ubiquitous hospital medication protocol. A key player in the molecular pathogenesis of HIT is the endogenous protein Platelet Factor 4 (PF4, CXCL4), which displays immunogenic neoepitopes upon complexation with heparin. Ensuing large immune complex formation with accommodation of multiple “HIT” IgG leads to platelet activation and thrombosis characteristic of HIT. It is thought that the auto-immunogenesis may be a side effect of PF4’s capacity to bind most Gram-negative bacteria (via lipid A), mediating adaptive and non-specific immunity.

 

This study explores the conformational changes in PF4 responsible for neoepitope exposure. Using hydrogen-deuterium exchange (HDX) coupled to online liquid chromatography-mass spectrometry (LC-MS), a kinetic profile can be built for both the whole protein via “global” exchange and at the peptide level via “local” exchange procedures. Analysis of the HDX profile allows deconvolution of distinct conformers of PF4 over ionic strength (0.15 – 1.5 M) and complex conditions (PF4 alone, PF4/heparin, PF4/lipid A if time allows). Additionally, local HDX experiments allow assignment of conformational behavior to specific segments of PF4.

 

Previous work has included binding assays, alanine scanning, circular dichroism, and native mass spectrometry to characterize the PF4/heparin complex and its behavior. However, none of the available studies offer significant insight into the distinct conformational states sampled or occupied by PF4 under HIT-like conditions. HDX-MS will add resolution of conformational populations and offer tertiary structural data to the knowledgebase surrounding HIT, and potentially elucidate its molecular mechanism.

Keywords

chemistry, biochemistry, mass spetrometry, blood, heparin

Research Area

Chemistry and Materials Science

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