The overall goal of this procedure is to apply the cook's kinetic method to determine the gas phase acidity of ine poly allin peptide. This is accomplished by first generating proton bound cluster ions. The second step is to perform the collision induced dissociation bracketing experiment.
Next, the selected reaction monitoring experiment is performed. The final step is to analyze the data collected from the selected reaction monitoring experiments. Ultimately, the kinetic plot is used to generate the value of the gas phase acidity.
The main advantage of this technique over conventional methods like titration experiments conducted in a solution is that sovereign effects can be eliminated. This method can help answer key questions in the field of bioorganic chemistry, such as how acidic is a peptide in a solvent free environment. The implication of this technique extended towards diagnosis of effective acid based properties of amino acid residues located in the interior of proteins because solvent effect are eliminated.
Generally individuals new to this method will struggle because the optimal mass spectrometry conditions are difficult to master without visual demonstration. Those Method can provide insight into the intrinsic acid-based properties of peptides. It can very well be applied to other systems such as peptide mimicking polymers and oligonucleotides.
We first had the idea for this method when we wanted to study confirmational effects on the acid-based properties of helico peptides. Begin this protocol with preparation of sample solutions for the cystine poly allin peptide and six halogenated carboxylic acids as described in the written protocol accompanying this video, the first step of the mass spectrometry measurement is to generate stable proton bound cluster ions of the peptide with a reference acid. Set the instrument in the negative ion MS mode with the electrospray ionization needle voltage at minus 4.5 kilovolts the capillary voltage at about minus 35 volts and the drying gas temperature to maintain at 150 degrees Celsius.
Set the first and third quadruple or Q1 and Q3 peak width to the calibrated scale. The peak width is an instrument parameter that can be used to adjust the resolution of the peaks. The calibrated scale setting allows display of narrow peaks for better peak separation.
The capillary voltage and the drying gas temperature can be adjusted. To enhance the observed ion abundances infuse about 0.5 milliliters of the sample solution of di fluoro acetic acid peptide into a one milliliter Hamilton syringe and connect the syringe to the electro spray ionization or ESI needle inlet using peak tubing. The syringe is then placed onto the syringe pump.
Turn on the syringe pump to infuse the sample solution into the ESI needle with the flow rate at 10 microliters per minute. Turn on the ESI needle voltage to activate the ESI process. Then turn on the detector.
A mass spectrum display should be observed in the profile mode. If the display is in the OID mode, switch to the profile mode. Watch the proton bound cluster ion formation by monitoring the peak at MZ 4 28.
Change the peak width from the calibrated scale to the 1.5 scale. The signal abundance of the cluster ion can be adjusted by fine tuning the instrument. Change the capillary voltage from minus 35 to minus 25 volts to maximize the abundance of the peak at MOZ 4 28.
The next step is to perform the collision induced dissociation or CID bracketing experiment. Once the abundance of the cluster ion reaches the desired value around 100 millivolts, switch the instrument to the MSMS mode. In this mode Q1 functions as a mass filter to isolate the cluster ion Q2 functions as the collision cell and Q3 functions.
As the mass analyzer set the collision gas pressure at 0.5 milour and the collision energy at 17 electron volts. Three peaks should be observed in the mass spectrum display window. The peak at M over Z 4 28 corresponds to the cluster ion.
The two peaks at M over Z 3 32 and M over Z 95 correspond to the deprotonated peptide and the deprotonated di fluoro acetic acid respectively. The minor peak at M over Z 2 98 is a secondary fragment from the deprotonated peptide. Acquire a CID spectrum for two minutes, perform similar CID experiments, and acquire a CID spectrum for the sample solution of the peptide with promo acetic acid.
Then perform similar CID experiments and acquire the CID spectra for the sample solutions of the peptide. With all other reference acids, the resulting CID spectra will be qualitatively similar, but the mass overcharge values and the relative peak heights will be different. The last step is to acquire the selected reaction monitoring or SRM spectra.
Switch the spectrum display to OID and set the instrument in the SRM mode. Keep the collision gas pressure at 0.5 milour. Keep M over Z 4 28 as the isolated ion by the first quadripole and fill in four M over Z values to be monitored by the third quadruple filling in each M over Z value four times the four MZ values are 95 for deproteinated di fluoro acetic acid 2 98 for the fragment of the peptide ion 3 32 for the pep peptide ion and 4 28 for the cluster ion.
Then change the polarity from positive to negative. Set the collision energy to four different values of electron volts, including 11.7, 17.6, 23.4, and 29.3. The set of the voltages is repeated four times for the corresponding ions to be monitored by the third quadruple.
Acquire the spectra for five minutes, perform similar measurements for the peptide with all other reference acids. Copy the values of the ion intensities from all the SRM spectra onto an Excel worksheet. Calculate the CID product ion branching ratios measured for all six proton bound clusters at all.
Four collision energies. Plot the values of branching ratio against the difference in gas phase acidity values. This will give four plots corresponding to the data.
At four collision energies extract the values of the slopes and the intercepts by linear regression of the four plots. In this case, the slopes are positive values and the intercepts are negative values. Give the symbol X to the slopes and the symbol Y to the intercepts.
Multiply the values of Y by negative one and use the symbol Y prime to represent positive values in order to allow the Y axis to display positive values. This conversion is optional as long as the corresponding values are used to make the plot for the next step plot the values of Y prime against the values of x. Linear regression of the plot yields a slope of 1.706 and an intercept of negative 0.536.
The slope corresponds to the difference in gas phase acidity. The value of average gas phase acidity is known to be 330.5 kcals per mole, which is determined by the set of the selected reference acids. The value of the gas phase acidity of the peptide is then obtained from the slope.
The CID bracketing experiments provide information on the relative acidities of the peptide compared to the selected reference acids two, representative CID spectra of the cysteine poly allin peptide with two reference acids. Dilu acetic acid and bromo acetic acid are shown here in this spectrum. The ion abundance represented by the peak height of the peptide ion is weaker than that of deprotonated di fluoro acetic acid.
Whereas in the other spectrum, the ion abundance of the peptide ion is stronger than that of deproteinated bromo acetic acid. The two spectra suggests that the gas phase acidity of the peptide is in the range between these two reference acids. The quantitative value of the gas phase acidity of the peptide is determined from the quantitative CID experiments.
The thermo kinetic plots for the dissociation of the proton bound clusters of the peptide with the six reference acids are shown here. Linear regression of the plots according to the thermo kinetic relationship between the gas VA acidity and the CID product ion branching ratio gives the value of the gas VA acidity of the cysteine poly allin peptide, which is 332.2 K cals per mole One method. This technique can be done in about five hours if you dis performed properly.
While attempting this method, it is important to remember to first check the performance of the mass spectrometer. One may perform an auto tune if it's necessary Following this procedure. Other methods like molecular modeling can be performed in order to answer additional questions like the most possible confirmations of the peptide After its development.
This technique paves the way for researchers in the field of mass spectrometry to explore a variety of molecular systems in terms of thermochemical properties. After watching this video, you should have a good understanding of how to use the mass spectrometry method to measure gas fat.Acidities. Don't forget that working with chemicals is extremely hazardous, and precautions such as wearing gloves and safety goggles should be taken while performing this procedure.
