Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry

Summary

Presented here is a protocol for a capillary electrophoresis-based hydrogen/deuterium exchange (HDX) approach coupled with top-down mass spectrometry. This approach characterizes the difference in higher-order structures between different protein species, including proteins in different states and different proteoforms, by conducting concurrent differential HDX and electrophoretic separation.

Abstract

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of protein therapeutics and the determination of the conformational transition pathways critical for biological functions, ranging from molecular recognition to enzymatic catalysis. Hydrogen/deuterium exchange (HDX) reaction coupled with top-down mass spectrometric (MS) analysis provides a means to characterize protein higher-order structures and dynamics in a conformer-specific manner. The conformational resolving power of this technique is highly dependent on the efficiencies of separating protein states at the intact protein level and minimizing the residual non-deuterated protic content during the HDX reactions.

Here we describe a capillary electrophoresis (CE)-based variant of the HDX MS approach that aims to improve the conformational resolution. In this approach, proteins undergo HDX reactions while migrating through a deuterated background electrolyte solution (BGE) during the capillary electrophoretic separation. Different protein states or proteoforms that coexist in solution can be efficiently separated based on their differing charge-to-size ratios. The difference in electrophoretic mobility between proteins and protic solvent molecules minimizes the residual non-deuterated solvent, resulting in a nearly complete deuterating environment during the HDX process. The flow-through microvial CE-MS interface allows efficient electrospray ionization of the eluted protein species following a rapid mixing with the quenching and denaturing modifier solution at the outlet of the sprayer. The online top-down MS analysis measures the global deuteration level of the eluted intact protein species, and subsequently, the deuteration of their gas-phase fragments. This paper demonstrates this approach in differential HDX for systems, including the natural protein variants coexisting in milk.

Introduction

Distinguishing protein species in different conformational, binding, or modification states and characterizing their structural differences are important for monitoring the pathways of transitions between these species involved in biological events, ranging from molecular recognition to enzymatic catalysis, and understanding the mechanisms underlying these events. Conventional biophysical techniques do not provide a complete solution due to the limitations such as insufficient resolution and loss of dynamic information in solution. Hydrogen/deuterium exchange coupled with mass spectrometry (HDX MS) is a technique that labels the structural and conformational features of proteins with deuterium (²H) via the exchange between labile hydrogen atoms of proteins and ²H from the deliberately introduced ²H₂O solution. Protons involved in hydrogen bonding or that are sequestered from the solvent in the protein interior do not exchange readily¹. Thus, as the exchange rate at an exchangeable site is highly dependent on its involvement in higher-order structures, the protein structures can be revealed at high spatial resolution by MS that probes the extent and rate of ²H-uptake based on the differing atomic masses between ¹H and ²H. Over the recent decades, HDX MS has become an outstandingly successful technique for studying protein conformations and dynamics².

In the classical bottom-up approach of HDX MS, the ensemble of protein species in different conformational, binding, or modification states is proteolyzed without separation at the intact protein level, making it infeasible to characterize individual species by analyzing the resulting proteolytic fragments with convoluted deuterium contents. In contrast, in the top-down approach, different protein states or proteoforms that have incorporated different deuterium contents give rise to multiple distributions of intact protein masses in an MS scan. This allows individual species to be separated by mass-selection of ions corresponding to each mass distribution using a proper mass filter (such as a quadrupole) and the characterization of their conformational differences in the subsequent tandem MS analysis^3,4,5,6. However, the efficiency of separating protein states or proteoforms in this strategy is limited by the extent of difference in their corresponding mass distributions.

Capillary electrophoresis (CE) provides a means to separate protein species based on their differing charges and hydrodynamic sizes in the solution phase with high efficiency⁷. Combining CE with HDX offers additional separation of protein states or proteoforms in the solution phase. In addition, the small volume of the CE capillary allows the utilization of a fully deuterated solution as the background electrolyte solution (BGE), i.e., the running buffer, rendering the capillary as an HDX reactor for protein samples. Due to the difference in electrophoretic mobility between proteins and protic reagents in the electrophoresis process, conducting HDX during CE results in a nearly complete deuterating environment for the protein analytes with minimal residual non-deuterated contents, thereby enhancing the sensitivity of the structural analysis using HDX data. As such, we developed a CE-based differential HDX approach coupled with top-down MS to characterize protein higher-order structures in a state- or proteoform-specific manner⁸.

This paper describes protocols for this approach by detailing the steps of material preparation, experimental procedure, and data analysis. Factors that may affect the method performance or data quality are listed in short notes. The representative results presented here include differential HDX data of mixtures of different proteins and natural variants of bovine β-lactoglobulin (β-lg), the major whey protein present in milk⁹. We demonstrate separation efficiency, reproducibility, and ²H-labeling performance of the two abundant variants of β-lg, i.e., A and B^10,11 during the CE-based HDX and variant-specific characterization of their conformations.

Protocol

NOTE: Use high-performance liquid chromatography (HPLC) grade or MS grade reagents whenever possible to minimize the contaminants that may interfere with MS analysis. Do not touch the CE-MS interface with bare hands during the measurement to avoid the possibility of an electrical shock caused by either the electrophoretic voltage or electrospray voltage. 1. Material preparation Modification of fused silica capillary for CE Prepare a 5% (w/w) hydroxypropyl cellulose (HPC…

Representative Results

Changing the infusion pressure of BGE allows the adjustment of both separation efficiency and migration time, which is equivalent to the HDX reaction time of the proteins to be separated (Figure 3). A lower infusion pressure results in better separation of CE peaks at the cost of the duration of the experiment (Figure 3A). A longer migration/HDX reaction time results in a higher level of deuteration of the protein analytes (Figure 3B</strong…

Discussion

The objectives of coating the inner wall of the CE capillary include the minimization of the electroosmotic flow and protein absorption during the CE process¹³. Although electroosmotic flow is beneficial for conventional CE analysis of small molecules owing to its capability of driving neutral or oppositely charged species to the detector, it compromises the separation efficiency of protein species with similar sizes and net charges in solution. Coating the capillary with HPC minimizes the electro…

Materials

ammonium acetate	Fisher Chemical	A/3446/50	≥99%
CESI 8000 plus capillary electrophoresis system	Sciex, USA
centrifuge	Eppendorf	5406000097
centrifugal filter	Merck	UFC201024	10 kDa cutoff
deuterium oxide	Energy Chemical	E090001	99.9 % D
formic acid	Acros Organics	270480250
fused silica glass capillary	Polymicro Technologies	1068150017	ID 50μm, OD 360μm
gas chromatography	Agilent	GC6890N
hydrochloric acid	Sigma Aldrich	258148
hydroxypropyl cellulose	Aladdin	H113415	MW 100000
magnetic stirrers	DLAB	8030101212
methanol	Fisher Chemical	A456-4	MS grade
microvolume UV-Vis spectrophotometer	DeNovix	84677JK7731
myoglobin	Sigma Aldrich	M1882
Orbitrap Fusion Lumos mass spectrometer	Thermo Fisher Scientific, USA
PA 800 Plus Pharmaceutical Analysis CE System	Beckman Coulter, USA
Q Exactive UHMR mass Spectrometer	Thermo Fisher Scientific, Germany
sodium hydroxide	Sigma Aldrich	S5881
ubiquitin	Sigma Aldrich	U6253
ultrasonicator	SCIENTZ	SB-5200
β-lactoglobulin	Sigma Aldrich	L0130