This experiment demonstrates how to utilize mass spectrometry to monitor hydrogen deuterium exchange associated with protein. Structural changes that lead to activation states first Cleve protein kinase PAC two with caspase three and then activate PAC two by auto phosphorylation using a TP as a substrate as a second step digest PAC two with Pepin and analyze the Prote PAC two fragments by tandem mass spectrometry proceed to analyze hydrogen deuterium exchange patterns of PAC two by mold off monitoring of the duration level of each PAC two fragment the precise level of hydrogen deuterium exchange using mold to analysis. Provide important insights into caspase three cleavage and auto phosphorylation of PAC two.
This method can help answer questions of protein dynamics such as protein conformational changes, protein ligand interactions, and protein-protein interactions. The main advantage of this technique over existing methods like HD exchange LCQ mass spectrometry is instrumental. This procedure does not require an HPLC linked mass spectrometry to analyze HD exchange results, and the procedure is easy to operate To cleave 0.6 micrograms of PAC two.
Add caspase three and incubate the reaction at 34 degrees Celsius for 30 minutes. Verify PAC two and complete cleavage of PAC two by analysis of products by SDS page. For the activation reaction, add the cleaved pack two to 20 microliters of activation buffer B and incubate at 34 degrees Celsius for 30 minutes.
Use SDS page to confirm auto phosphorylation by migration of the P 34 and P 27 fragments. First, wash the purine conjugated aro speeds twice with one milliliter of 0.1%Tri fluoro acetic acid dilute two microliters of 10 milligrams per milliliter. Pack two in 100 microliters of 0.1%TFA add 30 microliters of washed Pepsi in conjugated agro beads and incubate for five minutes.
Proceed to equilibrate a reverse phase H-P-L-C-C 18 column with 0.1%TFA at pH 2.5. Now load the Pepin Digest and dilute using a linear 100 minute gradient. Collect fractions every two minutes after drying the HPLC fractions with a speed VAC reus.
Suspend the samples in five microliters of aceto nitrile and 0.1%TFA at pH 2.5. As an initial screen. Analyze each fraction for peptides by moldy to for all fractions containing peptides.
Identify the fragments by tandem mass spectrometry. Use MS product function in protein prospector to match daughter ions and the theoretical MZ ratios based on the primary sequence of PAC two. First equilibrate all solutions and reagents for hydrogen deuterium exchange.
Initiate the hydrogen deuterium exchange by adding 18 microliters of buffer D two O to two microliters of 10 milligrams per milliliter. Pack two, perform reactions in triplicate over a time course. Quench the hydrogen deuterium reaction with 180 microliters of ice cold 0.1%TFA at pH 2.2.
Now distribute 100 microliter aliquots of hydrogen deuterium. Pack two to each 30 microliters of activated pepin conjugated aggregate beads. Incubate this pepin digestion on ice for five minutes.
Vortexing every 30 seconds effectively terminate the digestion reactions by centrifugation to remove the Pepin rapidly. Freeze the samples in liquid nitrogen for moldy to analysis. Prepare the matrix and store at zero degrees Celsius, partially thaw each sample quickly.
Mix one microliter of sample with one microliter of matrix, then spot on a four degrees Celsius moldy target plate 30 seconds prior to moldy to analysis. Apply a moderate vacuum to the plate to dry. The spots continue to acquire moldy to mass spectra average 100 scans in two minutes.
Proceed to use the Data Explorer 4.0 computer program to calculate the duration of each fragment. Then calculate the back exchange based on the ratio of the actual incorporated due to a number at 24 hours of hydrogen deuterium exchange divided by all possible exchangeable sites, which are the backbone amides, except the end terminal amide. In this example, CASP Bay's cleavage and auto phosphorylation are verified by SDS page Kumasi staining.
The non phosphorylated inactive PAC two is a single band of 58 kilodaltons CASP bay's. Cleavage of PAC two is complete and produces two fragments, P 27, the regulatory domain and P 34, the catalytic domain following auto phosphorylation. The migration of auto phosphorylated P 27 and P 34 shows an overlap on the protein gel due to the retarded migration of the fully phosphorylated P 27 fragment as compared to the non phosphorylated pack two.
This mass spectrum shows a time course of deuterium incorporation for inactive PAC two. The MZ peak 1697.85 is composed of multiple isotopic peaks as shown by the shift of the mass envelope. Over time, the red dotted line indicates the average of the mass envelope and the shift of the mass envelope shows the duration of a peptide Once mastered.
This technique can be completed in two days if it's performed properly. Don't forget that working with liquid nitrogen can be extremely hazardous, so take precautions such as poking a hole on the cap of the 1.5 mil EOL tube before putting it into liquid nitrogens.
