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# P kept constant at 50 pmole.

* Spiked standard (no spin column). B See Figure 2.26.

# P kept constant at 50 pmole.

* Spiked standard (no spin column). B See Figure 2.26.

Fig. 2.26 Plot of the DBQ (Drug, D)/CMVP A144L (Protein, P) molar ratios for the incubated concentrations as a function of the non-covalently bound concentrations as determined in the titration study of the GPC spin column eluates assayed by ESI-MS (see Table 2.5). The shape of the curve indicates that up to three drugs bind non-covalently and non-specifically to CMVP A144L.

Fig. 2.26 Plot of the DBQ (Drug, D)/CMVP A144L (Protein, P) molar ratios for the incubated concentrations as a function of the non-covalently bound concentrations as determined in the titration study of the GPC spin column eluates assayed by ESI-MS (see Table 2.5). The shape of the curve indicates that up to three drugs bind non-covalently and non-specifically to CMVP A144L.

phase H/D exchange methods have been developed to determine the stoichiome-try of the ligand-protein interaction by monitoring by ESI-MS the mass shifts of the protein undergoing H/D exchange upon titration with a ligand [32-34] (see Chapter 11).

2.3.3.3 Obtaining MS EC50s and Kds for Ligands Non-covalently Bound to Protein Active Sites

In biological systems, the concentration needed to inhibit 50% of a cellular reaction is called the IC50 for that reaction and is often obtained by titrating a given reagent with a fixed biological system. Similarly, a spin-column mass spectral (MS) EC50 (or Kd) can be obtained by analyzing the GPC spin-column ligand elu-ates from samples that were titrated with various concentrations with a fixed amount of protein. The mass spectral response for the ligand that non-covalently bound to the protein should produce a sigmoidal response curve and the concentration corresponding to 50% response from the extremes in the sigmoidal plot corresponds to the MS EC50. The EC50 value corresponds to the Kd value for the protein-ligand complex. (See the theoretical discussion in Section 2.1.4.4.) Recently, Schnier and coworkers [18] demonstrated this application of the GPC spin-column ESI-MS technique for obtaining the MS EC50s of a variety of compounds that non-covalently bind to a kinase protein. Figure 2.27 illustrates the

Fig. 2.27 GPC spin column ESI-MS determination of MS EC50S. Plot of fraction of known ligand inhibitor non-covalently bound to a fixed amount of kinase protein ([P]o, 5 mM) as a function of initial ligand concentration [L]o. The MS EC50 corresponds to the free ligand concentration [L] when 50% of the initial protein concentration is tied up as protein-ligand complex. At 50% of the

Fig. 2.27 GPC spin column ESI-MS determination of MS EC50S. Plot of fraction of known ligand inhibitor non-covalently bound to a fixed amount of kinase protein ([P]o, 5 mM) as a function of initial ligand concentration [L]o. The MS EC50 corresponds to the free ligand concentration [L] when 50% of the initial protein concentration is tied up as protein-ligand complex. At 50% of the normalized protein-ligand concentration, the [L]o value is read off the figure as 20 mM. The corresponding free ligand concentration [L] is [L]o - [C], see Eq. (4), where [C] is [P]o/2.0 mM or 2.5 mM. The MS EC50 value is therefore 17.5 mM, reasonably consistent with the biological IC50 of 8 mM (see reference [18]).

data obtained from a GPC spin-column ESI-MS titration of a known kinase inhibitor as a function of mass spectral ligand response and initial ligand concentration for a fixed protein concentration. From the sigmoidal fit of the mass spectral data, the MS EC50 value was 17.5 mM while the measured biological IC50 kinase inhibitor value was 8 mM. Good correlations were reported for the mass spectral EC50 and biological IC50 values for a variety of inhibitors.

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