Natalia Gasilova1 and Alexis Nazabal1
- Chemical Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 803
Analyzing the effect of ligands on protein–protein interactions is important to better understand the cellular processes. In vitro characterization of these modulations remains challenging because of the drawbacks associated with the analysis of noncovalent interactions. To facilitate the analysis, stabilization of the protein complex by chemical cross-linking followed by High-Mass MALDI mass spectrometry is a recently developed method offering several advantages: No need for immobilization or special tags, the analysis is possible directly on wild-type protein complexes, no need for buffer exchange, large applicability range for any type of protein complex from 0 to 1,500 kDa. Using this method, we analyzed the effect of the inhibitors Nutlin-3a and Nutlin-3b on the protein complex MDM2-p53. Using this fast and sensitive method, the IC50 values of these inhibitors have been determined.
CovalX Technology Used (Click each option to learn more)
To prepare the samples, a vial containing p53 was thawed for 20 minutes on the bench and then 5 μg was dissolved in 40 μl 1 x dilution buffer to give a 2.9 μM solution. Another vial containing 10 μg of full-length human MDM2 was thawed for 20 minutes (vial contains 50 μl of a 3.6 μM MDM2 solution). Both vials were submitted to buffer exchange with 0.5 ml desalting columns and the bottom closure of the column was removed before the column was placed in a 1.5 ml microcentrifuge collection tube. The column was centrifuged at 1000 x g for 1 minute to remove storage solution before being equilibrated by adding 300 μl of the exchange buffer (5 mM sodium phosphate, 7.5 mM sodium chloride, pH 7.2, 0.05% sodium azide) to the top of the resin bed and centrifuging the column again at 1000 x g for 1 minute. The flow-through was discarded and the equilibration process was completed two more times before the top of the column was blotted to remove the excess liquid. The column was moved to a new collection tube and 40 μl of the p53 is applied to the top of the now compact resin bed before being centrifuged at 1000 x g for 2 minutes. The same procedure was done with 40 μl of the MDM2 sample.
5 aliquots of the p53 solution were pipetted into 8 microcentrifuge vials and 20 μl of the MDM2 solution was pipetted into a separate 0.5 ml microcentrifuge tube. 10 μl of the CovalX K100 Stabilization Kit cross-linking buffer (5 mM sodium phosphate, 7.5 mM sodium chloride, pH 7.2) was added into 7 more 0.5 ml microcentrifuge tubes. A dilution series was performed using 10 μl of the MDM2 solution mixed with 10 μl of the CovalX K100 Stabilization Kit cross-linking buffer in the initial tube. The dilutions went as follows: 1 (3.6 μM), 1/2 (1.8 μM), 1/4 (900 nM), 1/8 (450 nM), 1/16 (225 nM), 1/32 (112 nM), 1/64 (66 nM), and 1/128 (33 nM). 5 μ of each diluted solution was mixed with each of the tubes containing 5 μl of p53 giving the final concentrations of p53/MDM2 (10 μl final): 1.4 μM/1.8 μM, 1.4 μM/900 nM, 1.4 μM//450 nM, 1.4 μM/225 nM, 1.4 μM/112 nM, 1.4 μM/66 nM, 1.4 μM/33, and 1.4 μM/16 nM.
For the control experiment, a matrix was created by dissolving 2 mg of sinapic acid (SA) in 200 μl of matrix buffer (acetonitrile:distilled water; 1:1, 0.1% TFA) to obtain a solution with a volume of 10 mg/ml and then the solution was sonicated at room temperature for 1 minute. 1 μl of each of the eight p53/MDM2 mixtures was mixed separately with 1 μl of the matrix before 1 μl of the final mixture of each was spotted on a MALDI plate. For the cross-link experiment, 2 mg of the CovalX R100 mixture (SBAT, SBBT, GBAT) was dissolved in 1 ml of DMF to obtain a solution with a volume of 2 mg/ml of cross-linker and then the solution was vortexed at room temperature for 1 minute. The remaining 9 μl of each of the eight mixtures from the control experiment were mixed with 1 μl of the cross-linking solution before being incubated at room temperature for varying times (20, 40, 60, 120, 180, 360 minutes) and mixed with 1 μl of the matrix. 1 μl of the final mixture was spotted on a MALDI plate.
The plates were then analyzed using a mass spectrometer that had been modified a CovalX HM2 detection system. The data obtained was analyzed using the CovalX Complex Tracker software. The effect of Nutlin-1 and Nutlin-3a (inhibitors) on the protein complex p53⋅MDM2 was analyzed using the target complex of High-Mass MALDI mass spectrometry.