Advantages of FPOP

One of the major advantages of using an irreversible reagent to probe protein interactions is that the sites of reaction can be readily determined by standard proteomic procedures. When the method is fully developed, one may be able to determine the Kd, binding stoichiometry, and the residues involved in ligand binding. As with PLIMSTEX, the hydroxyl radical method measures a change in the m/z and, therefore, is not susceptible to variations in ESI efficiency. With the ability to use any enzyme for digestion of the protein, FPOP can be used under any solution conditions including high salt, with denaturants and at low concentrations (we successfully applied this method to 100 nM protein). Current technology permits minute sample amounts to be used. In our experiments, approximately 50 pmol (800 ng) of apomyoglobin is loaded onto a protein trap. The sequencing stage requires only a few picomoles; thus, these experiments can be carried out on less than 80 ng of protein, a quantity that is less than the amount that can be observed on an SDS-PAGE gel using Coomassie staining. Given that analytical proteomic methods are being utilized, this method can be carried out in heterogeneous solutions, although multiple proteins in solution would complicate data analysis. Additionally, no specially labeled proteins are required, and this method, like PLIMSTEX, enables significant information to be gleaned from wild-type proteins; that is, no mutants are needed. Finally, although PLIMSTEX and other H/D exchange methods certainly have no significant effect on the equilibrium of the system, this method has not yet undergone significant testing to confirm the same property. FPOP, however, when properly carried out with suitable chemical scavengers in solution, should produce oxidized proteins that are modified before the protein complex can change conformation owing to the changes caused by modifying the protein or its substrate.

Finally, it is likely that this method can be adapted for high throughput drug screening studies. This may be particularly relevant for systems where activity assays cannot be readily developed. Conceptually, the protein complex would be mixed with a compound designed to disrupt the interface, irradiated, and digested online with trypsin or other proteases. The resulting peptides would be collected on column and eluted with a fast gradient into the mass spectrometer. Having previously mapped the interface, one can predict those oxidations that indicate a disrupted interface and set up the instrument to monitor selected ions. When observed, data-dependent fragmentation would confirm that a particular residue was modified, confirming that the test compound disrupted the protein interaction.

Additional developmental milestones for FPOP include the demonstration of Kd determination and development of dose-dependent radical foot printing by using different scavengers and concentrations to vary and then quantify the loss of unmodified peptide signal and the increase of modified peptide. Further advances can be made by determining with certainty the reaction timescale. At the present time, our calculations are worst case scenarios, so the reaction with 20 mM glutamine may be complete significantly before 1 ms. Direct analysis of the reaction timescale may be performed using a tandem laser setup that records in time the signal of a probe molecule sensitive to hydroxyl radical attack, or by following the formation and reaction of various aromatic amino acid residues on the protein for a direct reading of the longest possible reaction duration. Finally, with significant resources now being directed towards locating inhibitors by using novel methodologies, FPOP may soon be used to identify new drugs and targets.

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