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

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.

This method allows the separation of coexisting protein species, including different conformers or variants and online characterization of their higher order structure differences to be completed in a single CEMS measurement. It utilizes the electrophoretic effect to provide a nearly 100%duration environment for the HDX reactions and orthogonal separation of the coexisting protein species during HDX for top down MS analysis. Begin with preconditioning of the capillary electrophoresis, hydrogen deuterium exchange or CE HDX setup.

Using ultrasonication in a mixture of 50%methanol, 49%water and 1%formic acid, clean the flow through microvial CE-MS interface for at least 30 minutes at room temperature. Mount the HPC modified capillary on the capillary electropheresis or CE instrument. Rinse the capillary with background electrolyte solution or BGE for 10 minutes using the auto sampler and leave it filled with the BGE.

Use an appropriate length of unmodified fused silica capillary tubing as the infusion tubing for the modifier solution. Use a union and proper sleeve to connect the modifier tubing to a gas tight glass syringe with a blunt temp and rinse the tubing with the modifier solution for at least 10 minutes using an infusion pump. Insert the outlets of the HPC coded CE capillary and modifier tubing loaded with the corresponding solutions into the cleaned CE-MS interface.

Advance the syringe with the infusion pumps such that the modifier solution reaches the tip of the interface. Mount the assembled CE-MS interface on a nano electron spray ionization source housing of a mass spectrometer. Apply a spray voltage of 3-5 kilovolts to the CE-MS interface.

Infuse the modifier solution with the infuser pump at a flow rate ranging from 0.1-10 microliters per minute and ensure a stable electro spray at the tip of the CE-MS interface. Place the sample vial containing the BGE and the auto sampler, which will be used later to acquire blank electropherograms and blank mass spectra. Estimate the injection volume using the relationship between injection volume and injection parameters.

And inject the sample solution using the auto sampler at two PSI for an appropriate duration. Start the CE separation by applying an electrophoretic voltage of 20 kilovolts in an infusion pressure ranging from 0-2 PSI and acquire the electropherogram. Meanwhile, acquire the MS data in the chromatographic mode where the ion current graph is acquired as a function of time and the corresponding MS scans are not automatically combined into a single spectrum.

Save the blank electropherogram and mass spectra as references. Place the sample vials containing the desired concentrations of the protein sample solutions in the auto sampler and acquire the electropherograms and MS1 spectra of the electrophoretically separated and deuterium labeled protein species. Perform tandem Ms.measurements for the species of interest, either after acquiring the MS1 spectra within the same run or in a subsequent separate run.

After each measurement, flush the capillary with BGE at a pressure of 20 PSI for at least 10 minutes. Clean the CEMS interface and all tubing for storage upon completion of the experiments. Acquire a data set of the HDX endpoint sample with MS in direct infusion mode.

A low infusion pressure resulted in better separation of CE peaks, but led to an increase in both the migration time and duration of the experiment. A longer HDX reaction time resulted in a higher level of deduration of the protein analytes. The A and B variance of beta immunoglobulin or beta IG, which differ by only two amino acid residues in their sequence, gave rise to two adequately separated peaks in the EIC derived electropherogram.

The differential deuterium labeled variance resulted in a distinct mass distribution of ions. A lower number in relative abundance of Z ions than C ions was observed due to the unique dysofied linkage in confirmation of beta IG which limit the fragmentation efficiency between CS 82 and CS 176. Most of the fragment ions produced from beta IGA, and beta IGB exhibit a similar extent of deuterium uptake.

However, the larger segments covering the sequence variation sites from beta IGA were dedurated to a significantly greater extent than beta IGB. The pH of BGE is adjusted according to the isoelectric point of the sample to prevent protein aggregation and facilitate separation while maintaining the complex.