This work introduces an integrated microfluidic device for measuring rapid H/D exchange (HDX) in proteins. By monitoring backbone amide HDX on the millisecond to low second time scale, we are able to characterize conformational dynamics in weakly structured regions, such as loops and molten globule-like domains that are inaccessible in conventional HDX experiments. The device accommodates the entire MS-based HDX workflow on a single chip with residence times sufficiently small (ca. 8 s) that back-exchange is negligible (≤5%), even without cooling. Components include an adjustable position capillary mixer providing a variable-time labeling pulse, a static mixer for HDX quenching, a proteolytic microreactor for rapid protein digestion, and on-chip electrospray ionization (ESI). In the present work, we characterize device performance using three model systems, each illustrating a different application of 'time-resolved' HDX. Ubiquitin is used to illustrate a crude, high throughput structural analysis based on a single subsecond HDX time-point. In experiments using cytochrome c, we distinguish dynamic behavior in loops, establishing a link between flexibility and interactions with the heme prosthetic group. Finally, we localize an unusually high 'burst-phase' of HDX in the large tetrameric enzyme DAHP synthase to a 'molten globule-like' region surrounding the active site.
An electrospray coupled microfluidic reactor for the measurement of millisecond time-scale, solution phase kinetics is introduced. The device incorporates a simple two-channel design that is etched into polymethyl methacrylate (PMMA) by laser ablation. The outlet of the device is laser cut to a sharp tip, facilitating low dead volume 'on chip' electrospray. Fabrication is fast, straightforward and highly reproducible, supporting rapid prototyping and large-scale reproduction. Device performance is characterized using a cytochrome c unfolding reaction. Unfolding processes with rates in excess of 30 s Ϫ1 are easily measured, including the appearance of a 'native-like' intermediate that is maximally populated 180 ms post reaction initiation. To extract reliable rates from the data, a theoretical framework for the analysis of kinetics acquired under square-channel laminar flow is introduced. (J Am Soc Mass Spectrom 2009, 20, 124 -130)
A microfluidic reactor that enables rapid digestion of proteins prior to on-line analysis by electrospray ionization mass spectrometry (ESI-MS) is introduced. The device incorporates a wide (1.5 cm), shallow (10 microm) reactor 'well' that is functionalized with pepsin-agarose, a design that facilitates low-pressure operation and high clogging resistance. Electrospray ionization is carried out directly from a short metal capillary integrated into the chip outlet. Fabrication, involving laser ablation of polymethyl methacrylate (PMMA), is exceedingly straightforward and inexpensive. High sequence coverage spectra of myoglobin (Mb), ubiquitin (Ub) and bovine serum albumin (BSA) digests were obtained after <4 s of residence time in the reactor. Stress testing showed little loss of performance over approximately 2 h continuous use at high flow rates (30 microL/min). The device provides a convenient platform for a range of applications in proteomics and structural biology, i.e. to enable high-throughput workflows or to limit back-exchange in spatially resolved hydrogen/deuterium exchange (HDX) experiments.
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.
Many chemical and biochemical reactions equilibrate within a few seconds of initiation under "native" conditions. To understand the microscopic processes underlying these reactions, the most direct approach is to monitor the reaction as equilibrium is established (i.e. the reaction kinetics). However, this requires the ability to characterize the reaction mixture on the millisecond time-scale. In this review, we survey the contributions of time-resolved mass spectrometry (TR-MS) to the characterization of millisecond time-scale (bio)chemical processes, with a focus on biochemical applications. Compared to conventional time-resolved techniques, which use optical detection, the primary advantage of TR-MS is the ability to detect virtually all reactive species simultaneously. This provides a singularly high detail account of the reaction without the need for a chromophoric change on turnover or artificial chromophoric probes. To provide millisecond time-resolution, TR-MS set-ups usually employ continuous-flow rapid mixers, corresponding either to a fixed "tee" that provides a single reaction time-point or an adjustable position mixer that allows continuous reaction monitoring. TR-MS has been used to monitor processes with rates up to 500 s(-1) and to provide a detailed account of complex reactions involving 10 co- populated species. This corresponds to significantly lower time-resolution than optical methods, which can measure rates in excess of 900 s(-1) under ideal conditions, but substantially more detail (optical studies are typically limited to one or two analytes). TR-MS has been implemented on a number of platforms, including capillary and microfluidic set-ups. Capillary-based implementations are straightforward to fabricate and provide the most efficient rapid mixing. Microfluidic implementations have also been devised to enable multi-step experimental workflows that incorporate TR-MS. As a general method for time-resolved measurements, the applications for TR-MS are wide ranging. TR-MS has been used to identify intermediates in organic reactions, reveal protein (un)folding mechanisms, monitor enzyme catalysis in the pre-steady-state and, in conjunction with hydrogen-deuterium exchange, characterize protein conformational dynamics. While not without limitations, TR-MS represents a powerful alternative for measuring solution phase processes on the millisecond time-scale and a new, promising approach for revealing mechanistic details in (bio)chemical reactions.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.