Accelerated biomolecular kinetics revealed by microdroplet fusion mass spectrometry

Abstract

We have developed a new time-resolved mass spectrometry using fused microdroplets (PNAS, 2016). This technique enables (1) recording fast chemical reactions on the microsecond timescale which is about two orders of magnitude faster than previously developed time-resolved mass spectrometry, (2) capturing early molecular events occurring within a few microseconds, and (3) studying liquid-phase kinetics in confined environment such as in microdroplets which contrasts against the studies carried out in reactions in bulk solution. The power of this technique to monitor fast kinetics and to detect reaction intermediates was demonstrated through the kinetic studies of protein unfolding, the hydrogen-deuterium exchange in peptides, and protein-ligand interactions. We have found a markedly high acceleration of reaction rates in microdroplets by factors of 103 to 106 compared to bulk solutions regardless of reaction mechanisms, including specific covalent and nonspecific noncovalent bonding (Q Rev Biophys, 2015). To address the mechanism of the reaction acceleration, we examined several factors influencing the acceleration of acid-induced chlorophyll demetallation. The investigation of the effect of charge and the different solvent composition on the reaction rate acceleration revealed that the reaction acceleration was mainly attributed to the micro-confinement effect rather than the introduced charges, solvent composition, and solvent evaporation effect in microdroplet. The behavior of accelerated reaction suggests that the biochemical reactions occurring in confined environments such as in cells would significantly differ from what has been conventionally understood.