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Preparing Non-covalent Complexes in Protein Buffer; Protein Concentration, Ligand Concentration, Incubation Time

Non-covalent protein-drug complexes are prepared by incubating the drug with the native protein for 30-60 min in a compatible buffer. Volatile buffers are preferable over inorganic non-volatile buffers because mass spectral sensitivity is greater for samples prepared with volatile buffers. Often the libraries of drug candidates are prepared in DMSO solutions and are diluted with buffers similar to the ones used for the protein. The final DMSO concentration should be less than 5% so as not to denature the protein but to aid in solubilizing the drug candidates. Ideally, the drug candidates should be maintained in solution during the incubation process despite the fact that they often precipitate out of solution in the pH range normally utilized (pH 6-8) to maintain the protein in the native state.

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Fig. 2.5 Concentrations of staurosporine in eluate fractions obtained in sequential GPC spin column/HPLC ESI-MS experiments. Mixtures of compounds were spiked with staurosporine and incubated with PKA (-C-) and without PKA (-o-) as a control. The first eluate fraction shows the most significant ligand concentration difference between the protein-ligand and control samples. Reprinted from reference [16] with permission from Elsevier Science.

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Eluate volume [a*L]

Fig. 2.5 Concentrations of staurosporine in eluate fractions obtained in sequential GPC spin column/HPLC ESI-MS experiments. Mixtures of compounds were spiked with staurosporine and incubated with PKA (-C-) and without PKA (-o-) as a control. The first eluate fraction shows the most significant ligand concentration difference between the protein-ligand and control samples. Reprinted from reference [16] with permission from Elsevier Science.

The concentrations of the incubated protein and drug candidates used are a function of the outcome desired. If very strongly bound ligands to protein are desired (Kds < 0.1 mM) then lower concentrations of drug-protein mixtures are used; conversely, for weak binders (Kds > 10 mM) higher concentrations are used. For screening campaigns for moderate binders (Kds 0.1-10 mM) as well as strong binders using miniature spin columns of 100 mL volume with Tof mass spectrometer detectors, generally, protein concentrations of 5-10 mM with 5-10 times molar excess of drug candidates is sufficient. The volume of the protein-drug mixture utilized in the GPC spin column studies should be 10-15% of the miniature gel column volume. This small volume ratio is used so that the sample will not pass through open channels in the gel to the bottom of the column producing false positive results. Typically, 10 mL volumes of protein-drug mixtures are used with miniature spin columns of 100 mL volume. Screening studies for strong binders typically utilize protein concentrations of 0.25-5.0 mM with 1-5 times

Fig. 2.6 Titration assay of BSA and methyl orange (MO, MW 305 Da, Kd = 450 mM) by GPC spin column/ESI-MS methodology as a function of [MO]/[BSA] molar ratios in the positive ionization mode (A) and the negative ionization mode (B). The mass spectra in the top five panels are exploded views. The bottom panels of (A) and (B) illustrate the corresponding full spectra. The ion count for each spectrum is indicated in the upper right hand corner of each spectrum. A miniature 100 mL P6 GPC spin column was used to assay 10 mL samples.

BSA & MO Concentrations

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