Subsecond Time-Resolved Mass Spectrometry in Dynamic Structural Biology

Abstract

Life at the molecular level is a dynamic world, where the key playersproteins, oligonucleotides, lipids, and carbohydratesare in a perpetual state of structural flux, shifting rapidly between local minima on their conformational free energy landscapes. The techniques of classical structural biology, X-ray crystallography, structural NMR, and cryo-electron microscopy (cryo-EM), while capable of extraordinary structural resolution, are innately ill-suited to characterize biomolecules in their dynamically active states. Subsecond time-resolved mass spectrometry (MS) provides a unique window into the dynamic world of biological macromolecules, offering the capacity to directly monitor biochemical processes and conformational shifts with a structural dimension provided by the electrospray charge-state distribution, ion mobility, covalent labeling, or hydrogen–deuterium exchange. Over the past two decades, this suite of techniques has provided important insights into the inherently dynamic processes that drive function and pathogenesis in biological macromolecules, including (mis)­folding, complexation, aggregation, ligand binding, and enzyme catalysis, among others. This Review provides a comprehensive account of subsecond time-resolved MS and the advances it has enabled in dynamic structural biology, with an emphasis on insights into the dynamic drivers of protein function.

Apparatus for Automated Continuous Hydrogen Deuterium Exchange Mass Spectrometry Measurements from Milliseconds to Hours

“…The experimental rapidity of this technique is attributed to (i) the HDX short labeling times, (ii) automated replicate acquisition, and (iii) the lack of LC separation. We have noted in previous work that short (ms) HDX labeling times are sensitive to the types of interactions covered by conventional timescale HDX and also offer substantial advantages in characterizing disordered regions, weak binding interactions, and allosteric effects. , However, the inability to include LC separation in these experiments has been a substantial limitation, particularly since it has prevented the use of the nonvolatile buffers in which some proteins are maximally soluble. The “injection pulse” nature of millisecond discrete mode CFI-TRESI-HDX allows for automated online LC separation, which is carried out while the HDX module of the apparatus is being washed.…”

Electrospray Mass Spectrometry to Study Combinatorial iClick Reactions and Multiplexed Kinetics of [Ru(N3)(N∧N)(terpy)]PF6with Alkynes of Different Steric and Electronic Demand

“…In contrast, mass spectrometry (MS) has routinely been coupled to different sample injection techniques for decades and high-performance liquid chromatography (HPLC)/MS is now the method of choice for many analytical problems. A wide range of techniques has been developed for online and offline reaction monitoring using mass spectrometry, which extend to subsecond timescales, for example, to monitor dynamic processes in proteins . Furthermore, high-throughput reaction screening monitored by desorption electrospray ionization (DESI) mass spectrometry has recently been reported for several thousand organic reaction mixtures spotted by a robotic dispenser on a solid support .…”

Quinolinate Synthase: An Example of the Roles of the Second and Outer Coordination Spheres in Enzyme Catalysis

“…However, only in selected cases will it generate “snapshots” that are time-resolved enough to describe a whole catalytic reaction (although the development of “serial protein crystallography” is overcoming this difficulty). Other techniques, such as mass spectrometry, spectroscopic methods, and computational chemistry, can effectively be used in many cases.…”