An electrospray ms-coupled microfluidic device for sub-second hydrogen/deuterium exchange pulse-labelling reveals allosteric effects in enzyme inhibition

Abstract

In this work, we introduce an integrated, electrospray mass spectrometry-coupled microfluidic chip that supports the complete workflow for 'bottom up' hydrogen/deuterium exchange (HDX) pulse labelling experiments. HDX pulse labelling is used to measure structural changes in proteins that occur after the initiation of a reaction, most commonly folding. In the present case, we demonstrate the device on the β-lactamase enzyme TEM-1, identifying active site changes that occur upon acylation by a covalent inhibitor and subtle changes in conformational dynamics that occur away from the active site over a period of several second after the inhibitor is bound. Our results demonstrate the power of microfluidics-enabled sub-second HDX pulse labelling as a tool for studying allostery and show some intriguing correlations with mutagenesis studies.

HDX-MS guided drug discovery: small molecules and biopharmaceuticals

“…While the requirements for these approaches suggest that they are unlikely to become routine in the near future, combining newly developed HDX-MS compatible proteases may provide a feasible strategy to improve spatial resolution through subtractive analysis. Efforts to advance the detection limits of HDX-MS to sub-second time scales for the purpose of interrogating rapidly exchanging systems have been achieved through the development of micro-fluidic chips [70-72], as well as simulated approaches compatible with automated platforms [73]. …”

Utilizing Microchip Capillary Electrophoresis Electrospray Ionization for Hydrogen Exchange Mass Spectrometry

“…Furthermore, microfluidic technology is extremely well suited towards integration of multiple functional elements, 20,21 such as online digestion and peptide concentration, resulting in the prospect of a fully integrated microchip CE-ESI system for HX MS analysis. 22 …”

Changes in Signal Transducer and Activator of Transcription 3 (STAT3) Dynamics Induced by Complexation with Pharmacological Inhibitors of Src Homology 2 (SH2) Domain Dimerization

“…Unlike these static images, dynamic representations of protein structures can offer unparalleled mechanistic insights into biological processes and interactions by depicting the time evolution of protein conformational ensembles (15). Hydrogen-deuterium exchange (HDX) mass spectrometry (MS) when coupled with microfluidic sample processing enables the interrogation of protein dynamics of the native protein structures in solution, permitting the in situ observation of enzyme-catalyzed reactions (16) and protein-ligand interactions (17). HDX implemented on a microfluidic device for rapid mixing, quenching, and proteolytic digestion of the protein analyte offers substantial advantages over other techniques, including easy implementation, unlimited protein analyte size, time scales compatible with protein breathing motions, and site-specific resolution of up to a few amino acids (18).…”