A Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Platform for Investigating Peptide Biosynthetic Enzymes

This protocol can provide specially resolved information about protein conformational dynamics. Which can help to identify functionally important regions in protein of interest. This technique probes protein conformational dynamic under near-native conditions.

Without the need for protein labeling using nanomolar amount of material for each reaction. Begin by preparing undeuterated reference samples for the protein of interest in triplicate, in 500 microliter tubes. Quench the samples with the appropriate volume of quench buffer per tube to achieve a pH meter reading of 2.5.

Flash freeze the samples in liquid nitrogen for storage at minus 80 degrees Celsius until their analysis. Then analyze the protiated enzyme reference samples using a bottom-up liquid chromatography mass spectrometry workflow according to standard protocols. To analyze the raw mass spectrometry data in the appropriate Proteomic Software Program.

Navigate to Libraries and Protein Sequence Databanks to import the amino acid sequence of the protein of interest. Enter a name for the protein sequence of interest and import the protein sequence in FASTA format. To define the processing parameters under the Library menu, select Electrospray MSE as the data acquisition type.

In the Lock Mass for Charge 2 field, enter 785.8426 for the master charge ratio for the two plus ion of glucan one fiber no peptide B and click Finish. To define the workflow parameters, select Electrospray MSE for the search type. Under the Workflow and Database search query heading, select the database protein created in the Databank field and change the primary digest reagent to nonspecific.

To clear the fixed modifier reagent field, hold the control button while clicking Carbamidomethyl C.To specify the output directory, select Options, Automation Setup, and Identify E.Then check the apex 3D and peptide 3D output and ion accounting output boxes. And specify the desired directory. To process the referenced sample data, right-click on the microtiter plate to create a new plate.

Left-click in one well and drag to three other wells to highlight one well for each referenced sample. Next, right-click and select Add Raw Data. In the window, navigate to the directory containing the three referenced files and select all three files at the same time.

Click Next and select the defined processing parameters. Click Next again and select the defined workflow parameters. Then click Finish.

Once the raw data, processing parameters, and workflow parameters have been assigned to each well on the plate, the wells will turn blue. Select the wells, right-click and select Process Latest Raw Data. To track the processing of the data, click the right bottom corner of the window.

When no job to run appears, the processing is complete and the wells in the microtiter plate will turn green. Right-click on the wells and select View Workflow Results. A separate window will open for each referenced data file.

Inspect the data to ensure that the majority of the mass spectrometry signals in the referenced sample data were successfully mapped to the peptides predicted from the in silico digestion of the protein of interest. Matched peptides will appear blue in the output spectrum. Double-click the okay filter and check that the percent coverage is greater than 99%Import the Proteomic Software output into the Hydrogen Deuterium Exchange Processing Software for additional thresholding.

And click Data and import PLGS results. Click the Add icon and navigate to the appropriate directory to select the processed data files. Click Next and set the minimum consecutive ions to greater than or equal to two.

The mass error to five parts per million. And the file threshold to three, then click Finish. To conduct hydrogen deuterium exchange reactions, aliquot quench buffer in 500 microliter tubes.

And briefly centrifuge to transfer all of the quench buffer to the bottom of each tube. Place the tubes containing quenched solution on ice. Next, premix all of the deuterated reaction components in a separate 1.5 milliliter tube.

Then transfer the reaction mixture to a 25 degrees Celsius temperature controlled waterbath for a 10-minute incubation. Add enzyme to the deuterated reaction components to initiate the exchange reaction. Mix the reaction contents thoroughly by pipetting up and down.

At the appropriate experimental exchange time points remove 50 microliter aliquots from each hydrogen deuterium exchange reaction. And mix the reactions quickly and evenly with one aliquot of ice-cold quench buffer in individual 500 microliter tubes. Then immediately cap the tubes for flash-freezing in liquid nitrogen.

Store the samples at minus 80 degrees Celsius until ready for analysis. After thawing the frozen samples, analyze the deuterated enzyme reference samples using the same bottom-up liquid chromatography mass spec workflow developed for the undeuterated reference samples. After collecting the LCMS data for the deuterated samples, import the data into the previously created hydrogen deuterium exchange project for data processing.

And click New State and New Exposure to define the biochemical states and deuterium exposure times respectively pertinent to the analysis. Click New Raw to select the hydrogen deuterium exchange data files to be analyzed. And assign the appropriate exchange times and biochemical state to each raw data file that is imported.

For analysis and visualization of the data, select the first peptide in the Peptide list and open the stacked spectral plot from the Views menu. Click the mouse to assign and unassign sticks as necessary to ensure that the proper isotope distribution has been located in the data. And that each isotope peak has been assigned.

To check the stick assignments for each charge state toggle the charge state at the top of the stacked spectral plot window. To check the standard deviation of the peptide deuterium uptake values in the Views menu, select the coverage map. Which displays each peptide in the Peptide list mapped along the amino acid sequence of the protein of interest.

Color the peptides according to relative standard deviation and visually search the map for outlier peptides with the high relative standard deviation. Click the outlier peptides in the coverage map to populate the stacked spectral plots and the data viewer window with the target peptide. Display the difference of interest in the coverage map and right-click on the map to export the difference data to a CSV file.

Import the difference, and state data, and the PDB file of the protein of interest into Deuteros. And select the 99%confidence interval and Enable Sum and process the data. Then under pi mol select Export Uptake and Export to generate a PyMOL script to map the regions of significant exchange difference onto the PDB structure of the protein of interest in the PyMOL software.

These representative total ion chromatograms for three reference samples of Hal-M2 lambda peptide synthetase show the time-dependent changes in the sum of all of the ion counts at all of the mass-to-charge ratio values included in the mass spectral scan. The shape and intensity of the total ion chromatogram profile should be similar. Indicating that the proteolytic digestion of Hal-M2 is reproducible.

And is of similar efficiency in all three reference samples. The mass spectra over a given time interval should also be similar. Providing confidence that similar peptides are present within each sample and are alluding at similar times from the C18 column.

Most of the peptides in the deuterium exchanged samples should exhibit obvious shifts in there isotopic distributions toward higher mass-to-charge ratio values. For example, these data indicate that a substantial fraction of the deuterium label was being maintained throughout the course of the acid quench, pepsin digestion, and liquid chromatography mass spectrometry data collection. After characterization of the biochemical properties of the enzyme as demonstrated, representative hydrogen deuterium exchange mass spectrometry results such as these can be generated.

To illustrate the binding of the Hal-A2 precursor peptide to the Hal-M2 AMP, PMP complex. The hydrogen deuterium exchange reactions must be prepared and quenched in a consistent manner. And you must be methodical when determining the deuterium exchange values for numerous peptide detected.

Hydrogen deuterium exchange mass spectrometry provide peptide-level information and dynamic protein conformational changes. The functional relevance of any hydrogen deuterium exchange should be validated for Meter Genesis. And orthogonal activity assays.