From small-molecule reactions to protein folding: studying biochemical kinetics by stopped-flow electrospray mass spectrometry

This work introduces stopped-flow electrospray ionization (ESI) mass spectrometry (MS) as a method for studying fast biochemical reaction kinetics. After initiating a reaction by rapid mixing of two solutions, the mixture is transferred to a reaction vessel and a steady liquid flow to the ESI source of the mass spectrometer is established. The kinetics are studied in real time by monitoring selected ion intensities as a function of time. In order to characterize the performance of this setup the acid-induced demetallation of chlorophyll a was studied. It was found that the reaction is second order in acid concentration and that pseudo-first-order rate constants of up to roughly 7 s−1 can be measured reliably. Stopped-flow ESI MS was also applied to study the acid-induced denaturation of myoglobin. The data presented here confirm the occurrence of a short-lived unfolding intermediate during this reaction. Stopped-flow ESI MS can provide information that is not accessible by optical rapid-mixing experiments. Therefore it appears that this novel technique has the potential to become a standard tool for kinetic studies in a number of different fields.