All organisms have disorder in their proteome, estimated up to 33%These regions are observable by hydrogen/deuterium exchange mass spectrometry, but only in the millisecond time range. Our protocol will show you how to do this successfully. This is a fully automated analytical technique.
You just set everything up, program the desired time measurements, hit go and walk away. This technique opens up the exciting opportunity of screening for compounds that stabilize specific confirmations of intrinsically disordered proteins such alpha-synuclein. To begin sample preparation for peptide mapping, first take a vial of stored alpha-synuclein protein stock from the minus 80 degrees Celsius freezer.
And once it is thawed, filter using a 0.22 micrometer syringe filter. Then measure the absorbance of the solution at 218 nanometers to determine the protein concentration using Beer-Lambert's law, and dilute the protein with equilibrium buffer to a final concentration of five micromolar. Next, to set up the autosampler robot, add 50 microliters of the five micromolar protein solution to a total recovery vial, and place the vial in the sample position in the right chamber of the hydrogen/deuterium exchange, or HDX, robotics in line with the mass spectrometer.
Then put one vial of equilibrium buffer and two vials of pepsin wash buffer to reagent positions one, four, and five of the left chambers and one vial of quench buffer to reagent position one in the HDX right chamber and set the temperature at 20 degrees Celsius using the Peltier temperature controller. Then add an equal number of total recovery vials and maximum recovery vials in the same reaction positions of the HDX left chamber and HDX right chamber respectively. Next, set up a sample list with appropriate LC and MS methods in the scheduling software and start the schedule.
To obtain the final peptide coverage map, manually curate the isotopic assignments in the DynamX HDX data analysis software. After cleaning the Fast HDX prototype instrument, open the graphical user interface of the compatible HDX software to initialize the system. Enter 20 degrees Celsius and 0.5 degrees Celsius as the sample chamber and quench chamber temperatures respectively, followed by clicking set to apply the new temperatures.
To clean and prepare the instrument, set up the centrifuge tubes with LC/MS grade water at all inlets, then check both left and right syringes in the titrator plumbing delivery tab and continue clicking prime until all latent air bubbles in the tubes are gone. To check all the boxes for syringes, first go to the macros tab and click on calibrate syringes home position, then first click wash syringe load loop, followed by wash all mixing loop volumes. If there is any bubble in a buffer syringe, disconnect the syringe and degas it by vertically ejecting the bubble.
Before recalibrating to the zero position, replace the syringe. For setting up the Fast HDX prototype instrument for HDX experiments, first insert the left titration inlet tube into the total recovery vial. To prevent temperature-induced oligomerization and aggregation, place the tube in a tabletop fridge.
Next, add 50 milliliters of equilibrium, labeling, quench, and pepsin wash buffers to the respective buffer inlets in the right and left chambers of the instrument, followed by adding 50 milliliters of column wash buffer to the pepsin wash inlet. For priming the protein and column wash lines, check the right and left syringes in the titrator plumbing delivery tab and click prime once. After checking all the boxes for syringes as demonstrated earlier, go to the manual quench flow tab to enter the required settings.
For a time course experiment, use the symbolic dot button to enter the times in milliseconds. Then set the trap time to three and the wait for HPLC to trap time plus run time plus 1.5 minutes. If blank experiments are run between sample runs, click on the run blank box after ensuring an entry in the acquisition software for the blank run after each sample run.
Once the sample list is ready and the appropriate entries have been highlighted, start the run by clicking play and Fast HDX in the software. For data processing, open the file menu of the file of spectrally assigned peptides from the peptide mapping experiments and click open in the DynamX software, then import the raw files to the DynamX software. After opening the data menu, click on MS Files.
To create states for each protein condition being studied, click on new state. To add each HDX time point, click on new exposure, then click new raw. To import raw files, drag each file to the correct position and click OK when finished.
After automatically assigning isotopes, manually curate the isotopic assignment to ensure high data quality. Export the cluster data in csv file with the column order as described in the manuscript. Open the data menu in the mass measurement software and click on export cluster data.
For data analysis, first load the exported clustered data into the HDX analysis software HDfleX. To fit the experimental data for all experiments and states, select the appropriate back exchange correction methods to get the observed rate constants for the HDX reaction. Then calculate a global significance threshold using a preferred method in HDfleX and perform hybrid significance testing to determine the significant differences across the states compared.
The mapping experiment on alpha-synuclein generated a peptide coverage map that covered 100%of the protein sequence with an average redundancy of 3.79. Moreover, this protocol enabled the generation of deuterium uptake curves for different peptides of alpha-synuclein, which were then expressed as fitted and back exchange corrected graphs. It was evident that most hydrogen/deuterium exchange in alpha-synuclein was done by one second.
The conformational difference between states A and B of alpha-synuclein was also evident from the higher deuterium uptake by state B both at the peptide and amino acid levels. For such a sensitive technique, it is important to use robust statistical significance tests when comparing data. Here, we used our software HDfleX.
