In‐droplet hydrogen–deuterium exchange to examine protein/peptide solution conformer heterogeneity

Abstract

Rationale Many different structure analysis techniques are not capable of probing the heterogeneity of solution conformations. Here, we examine the ability of in‐droplet hydrogen–deuterium exchange (HDX) to directly probe solution conformer heterogeneity of a protein with mass spectrometry (MS) detection. Methods Two vibrating capillary vibrating sharp‐edge spray ionization (cVSSI) devices have been arranged such that they generate microdroplet plumes of the analyte and D2O reagent, which coalesce to form reaction droplets where HDX takes place in the solution environment. The native HDX–MS setup has been first explored for two model peptides that have distinct structural compositions in solution. The effectiveness of the multidevice cVSSI–HDX in illustrating structural details has been further exploited to investigate coexisting solution‐phase conformations of the protein ubiquitin. Results In‐droplet HDX reveals decreased backbone exchange for a model peptide that has a greater helix‐forming propensity. Differences in intrinsic rates of the alanine and serine residues may account for much of the observed protection. The data allow the first estimates of backbone exchange rates for peptides undergoing in‐droplet HDX. That said, the approach may hold greater potential for investigating the tertiary structure and structural transitions of proteins. For ubiquitin protein, HDX reactivity differences suggest that multiple conformers are present in native solutions. The addition of methanol to buffered aqueous solutions of ubiquitin results in increased populations of solution conformers of higher reactivity. Data analysis suggests that partially folded conformers such as the A‐state of ubiquitin increase with methanol content; the native state may be preserved to a limited degree even under stronger denaturation conditions. Conclusion The deuterium uptake after in‐droplet HDX has been observed to correspond to some degree with peptide backbone hydrogen protection based on differences in intrinsic rates of exchange. The presence of coexisting protein solution structures under native and denaturing solution conditions has been distinguished by the isotopic distributions of deuterated ubiquitin ions.

Effect of pH on In-Electrospray Hydrogen/Deuterium Exchange of Carbohydrates and Peptides

“…Amides have a minimum exchange rate around pH 2.5. Yet, because amides experience slower rates of exchange, on the time scale of milliseconds to seconds in unstructured environments, , we expect minimal labeling for amides during ESI . Amines exchange on a time scale similar to hydroxyls, between microseconds and milliseconds, but amines have a consistent, minimum exchange rate at or below pH 4 .…”

“…Yet, because amides experience slower rates of exchange, on the time scale of milliseconds to seconds in unstructured environments, 32,38 we expect minimal labeling for amides during ESI. 52 Amines exchange on a time scale similar to hydroxyls, between microseconds and milliseconds, but amines have a consistent, minimum exchange rate at or below pH 4. 32 For Substance P, the D uptake trend is similar to that expected based on the bulk-solution exchange rates of amines, with the lowest D uptake at pH 3 and increasing D uptake for pH 8 and 10.5.…”

A millimeter water-in-oil droplet as an alternative back exchange prevention strategy for hydrogen/deuterium exchange mass spectrometry of peptides/proteins

“…In contrast to the millimeter droplet, HDX is reported to be accelerated in the ESI micrometer droplet via chemical pathways that are inaccessible to the millimeter droplet. 28–30 For instance, the high voltage applied in an ESI experiment causes an excess charge distribution near the droplet surface, resulting in a radial distribution of charge within a single ESI droplet. 31 Due to the small size of the ESI micrometer droplet, this surficial charge distribution induces a strong electric field that covers a large volume of the droplet.…”

Structure Characterization of an Intrinsically Disordered Peptide Using In-Droplet Hydrogen Deuterium Exchange Mass Spectrometry and Molecular Dynamics Simulations

“…As mentioned above, HDX-MS is being developed as a novel characterization and structure determination technique to provide conformational dynamics and distinguishing of co-existing structures information for proteins in the solution phase. 48 In this work, in-droplet HDX-MS provides similar dynamics information over a very brief (µs) timescale to enable the capture of local folding/unfolding events. The utilization of MD simulations confirms the conformational information from the HDX-MS experiments (relative number of deuterium incorporation) through the creation of a reaction model.…”

“…The ionization approach has been further demonstrated for distinguishing different structures using a multidevice approach where rapid HDX reactions occur through the process of droplet mixing on the millisecond and sub-millisecond timescale. 44,47,48 Notably in-droplet HDX is shown as a means to achieve exchange reactions that are decades faster than the standard bulk solution approach. 49 This approach is already shown to distinguish coexisting structures of proteins in solution as well as elements of peptide secondary structure.…”