The overall goal of the following experiment is to analyze the confirmational flexibility in proteins and changes thereof in response to environment, ligand binding or protein protein interactions. This is achieved by pre incubating the protein of interest under different conditions which are suspected to influence the confirmation of the protein, such as ligand binding. The reaction mixture is then diluted into deuterium oxide, incubated for different time intervals, and subsequently analyzed by liquid chromatography mass spectrometry to determine the degree of deutero incorporation into the peptide backbone.
Amide groups, because protected areas have decreased incorporation of deuteros, comparing the peptide spectra of the experiment in the absence and presence of a binding partner reveals a shift of the OID of the spectra due to the weight difference between proton and deuteron. Regions that exhibit prominent protection after addition of a binding partner are potential binding sites. Depending on the sequence coverage and the availability of overlapping peptides, binding sites can be narrowed down to a few amino acids.
This method can help to answer key questions in the protein folding and the protein dynamics field, such as how proteins fold, how they unfold, how they react to physical and chemical impacts like temperature increase or heavy metals. How likened binding and protein-protein interactions can influence the confirmation of a protein. It is also very important for the development of protein drugs for verification of proper folding and for general quality control.
Generally, individuals new to this method may encounter several difficulties. If the protein, for example, is not well digested by pepsin, the sequence coverage will be low. If the protein is very aggregation prone, it may aggregate during incubation or after adding the quench buffer and will result in low signal intensities in the mass spectrometer.
In this demonstration, We will investigate the binding of the yeast molecular chaperone, HP 90 to its cos chaperone Sty one using hydrogen exchange mass spectrometry STY one binds to HP 90 and facilitates client binding by inhibition of HB ninety's ABAs activity. Begin this procedure with preparation of buffers, samples and beads as detailed in the text protocol. To prepare the columns for amide hydrogen exchange, unscrew one side of the guard column and remove the filter tightly.
Screw the packing funnel onto the open end of the column. Use a 16th inch adapter to attach an empty five milliliter syringe to the bottom outlet of the column. Make sure to fix it gas tight to the guard column.
Apply a few drops of slurry bead material on top of the funnel. Pull the plunger of the syringe to aspirate the slurry through the funnel into the guard column. Apply more slurry bead material onto the funnel and continue the procedure until the guard column is completely filled with bead material.
Then remove the funnel before placing the filter and filter ring onto the open end tightly. Screw the column cap onto the guard column and remove the syringe from the other side. Close both ends of the guard columns with plugs.
To avoid drying out of column material to set up the system for hydrogen exchange mass spectrometry. First connect the trap column to the high performance liquid chromatography or HPLC system. Equilibrate the column by setting the flow rate of pump A to 0.4 milliliters per minute with 0.1%formic acid as the solvent.
After calibrating the mass spectrometer, connect the outlet of the HPLC to the source of the mass spectrometer. To determine the peptic peptides first, connect the Pepin column and then the analytical column to the system. Set up the parameters for chromatography and mass spectrometry in the control software by choosing the gradient type and mass spectrometry method.
Choose a long gradient to ensure good chromatographic resolution. Enable tandem mass spectra on the mass spectrometer. Prepare 100 to 200 pomo of heat shock protein 90 or HSP 90 in 100 microliters of H two L buffer.
Add 100 microliters of quench buffer and mix by pipetting up and down. Inject the 200 microliters sample into the injection port of the injection valve with a Hamilton syringe. Switch the injection valve into the inject position and start the chromatography program.
Next, identify peptic peptides of heat shock protein 90 by searching a database for the resulting peptides. Decrease the system temperature to zero degrees Celsius by adding ice water. Repeat this step without tandem mass spectrometry and with the gradient that will be used for the actual hydrogen exchange experiment.
For heat shock protein 90 and STI one, use a 10 minute gradient from 90%solvent, a 10%solvent B to 45%solvent, A 55%solvent B, determine the retention times of the identified peptic peptides in the gradient used and create a list comprising peptide sequence, peptide charge, state, and retention time. This will be used to identify each peptide after hydrogen exchange experiments. To identify protein protein interaction interfaces, first set up the gradient and mass spectrometry method.
In the control software, use a 10 minute linear gradient from 90%solvent, a 10%solvent B to 45%solvent. A 55%solvent B load a mass spectrometry method that is optimized for detection between 300 to 1, 500 mass to charge ratios, although most of the peptides will be below the 1000 mass to charge ratio. Set the injection valve into load position and the six port valve.
In loading desalting position, set the flow rate of the loading pump to 0.4 milliliters per minute. After running the unchanged reference and the 100%control sample as described in the text protocol, prepare 20 to 100 pico mole of heat shock protein 90 in a volume of one to five microliters. Add temperature adjusted deuterium oxide buffer to bring the sample volume up to 100 microliters incubate for the exact period of time that was previously defined as described in the text protocol.
When determining the dynamic range of exchange, then add 100 microliters of ice cold, quench buffer, and pipette up and down twice quickly inject the 200 microliters into the injection valve of the HPLC. Switch the injection valve to the inject position and immediately start the chromatography program. After two minutes, switch the six port valve from the desalt loading position to the elute position.
Do this for each protein individually to determine the deuteron incorporation into each peptide in the absence of the interacting protein to determine the interaction surface mix heat shock protein 90 with an at least twofold excess of STI one to shift the equilibrium to the bound state, incubated the desired temperature until complex formation is at equilibrium. Following incubation, add temperature adjusted deuterium oxide buffer to bring the sample volume up to 100 microliters and incubate for exactly a defined period of time. Then add 100 microliters of ice cold quench buffer, and pipette up and down twice quickly inject the protein and run as before.
Analyze the acquired data with suitable software and use the determined retention times to find each peptide in the analysis. Calculate the OID of the isotope distribution for the unchanged protein and for the hydrogen exchange experiments, compare the incorporation of deuteros of target protein alone and with excess of binding partner. This can be done automatically with commercial software or manually with the spreadsheet program M, the direct protein protein interaction between HS P 90 and STI one was tested by comparing the STI one peptide spectra in the absence and presence HS P 90 following deuterium oxide labeling.
The resulting difference plot reveals that mostly peptides in TPR two A and TPR two B show a strong protection in the presence of HS P 90, indicating that these regions interact with HS P 90. The protection in TPR two A can easily be rationalized from the crystal structure of STY one TPR two A TPR two B in complex with an HS P 90 peptide, which is colored according to the number of amyloid protons protected from DEUTERO incorporation. Such a clear protection in protein protein interactions is not always observed.
The protection in HSP 90 in the presence of STI one is not as pronounced and not as localized as in STI one. All peptides show slightly increased protection from deutero incorporation. Shown here is a cartoon representation of yeast HS P 90 colored according to the number of amli protons protected from deuteron incorporation, STY one stabilizes HS P 90 globally as no region of the protein shows more flexibility in its presence.
It is not clear if this reduced deuteron incorporation originates from direct interaction with sty one or through allosteric effects. On the confirmation of HSP 90 continuous labeling, hydrogen exchange, mass spectrometry was used to study the dynamics of different protein regions. Shown here are the spectra of peptide 43 to 62 from the nucleotide binding domain of HSP 90.
In the presence and absence of STI one, the peptide spectra show a time dependent incorporation of deuterium that increases with longer incubation. The degree of protection changes throughout the incubation time, showing greater difference at the shorter incubation times and almost similar exchange rates at the longest time point. This suggests a lower degree of stabilization or a region of dynamic interactions with frequent dissociation and ssociation.
While attempting this procedure, it is important to remember to keep conditions meticulously constant and to have everything ready before starting the exchange reaction. Following this Procedure, other methods can be used to answer additional questions like the location of interaction sites in protein complexes.
