Dissociation of the TNF-alpha trimer caused by the small-molecule inhibitor SPD304 was monitored using native ESI-MS and ion mobility spectrometry. Upon addition of inhibitor, our data clearly indicates partial dissociation of the protein into dimers and monomers. The IMS-MS analysis shows that dimeric ions have their own characteristic drift time distributions, which are different from those of the dimer ions originating in the gas phase due to CID. We show that only one equivalent of the inhibitor binds to the trimeric form. We also investigated inhibition of the heterodimer formation of the survival protein Bcl-xL and cell death-promoting regions of the proteins Bak and Bad, using the small inhibitors ABT737... More
Dissociation of the TNF-alpha trimer caused by the small-molecule inhibitor SPD304 was monitored using native ESI-MS and ion mobility spectrometry. Upon addition of inhibitor, our data clearly indicates partial dissociation of the protein into dimers and monomers. The IMS-MS analysis shows that dimeric ions have their own characteristic drift time distributions, which are different from those of the dimer ions originating in the gas phase due to CID. We show that only one equivalent of the inhibitor binds to the trimeric form. We also investigated inhibition of the heterodimer formation of the survival protein Bcl-xL and cell death-promoting regions of the proteins Bak and Bad, using the small inhibitors ABT737 and ABT263. We found that the ABT737 is more potent compared to ABT263 in preventing the heterodimerization between Bcl-xL with Bak and Bad derived BH3 peptide. We could also monitor the mode of binding, which in this case is competitive. These results indicate that native ESI-MS can be widely used to study the inhibition of other relevant protein-protein interactions (PPIs), and provide a good basis for further improvement and identification of small-molecules PPI inhibitors.
