title: Characterizing rapid, activity-linked conformational transitions in proteins via sub-second hydrogen deuterium exchange mass spectrometry.
abstract: This review outlines the application of time-resolved electrospray ionization mass spectrometry (TRESI-MS) and hydrogen-deuterium exchange (HDX) to study rapid, activity-linked conformational transitions in proteins. The method is implemented on a microfluidic chip which incorporates all sample-handling steps required for a 'bottom-up' HDX workflow: a capillary mixer for sub-second HDX labeling, a static mixer for HDX quenching, a microreactor for rapid protein digestion, and on-chip electrospray. By combining short HDX labeling pulses with rapid digestion, this approach provides a detailed characterization of the structural transitions that occur during protein folding, ligand binding, post-translational modification and catalytic turnover in enzymes. This broad spectrum of applications in areas largely inaccessible to conventional techniques means that microfluidics-enabled TRESI-MS/HDX is a unique and powerful approach for investigating the dynamic basis of protein function.
authors.affiliation: Department of Chemistry, York University, Toronto, Ontario, Canada.
authors.forename: Diana
authors.initials: D
authors.lastname: Resetca
authors.forename: Derek J
authors.initials: DJ
authors.lastname: Wilson
mesh_terms: D045304:Cytochromes c / Q000737:chemistry; D041961:Deuterium Exchange Measurement / Q000295:instrumentation / Q000379:methods*; D004798:Enzymes / Q000737:chemistry; D064267:Intrinsically Disordered Proteins / Q000737:chemistry; D046210:Microfluidic Analytical Techniques; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*
chemical_list: D004798:Enzymes; D064267:Intrinsically Disordered Proteins; D011506:Proteins; D045304:Cytochromes c
keywords: capillary mixer; conformational transitions; hydrogen/deuterium exchange; intrinsically disordered proteins; label-free analysis; microfluidics; pepsin digestion microreactor; protein dynamics; time-resolved electrospray ionization mass spectrometry; ‘bottom-up’ HDX workflow