Comparing Noncovalent Binding of Ligand to Mutated Proteins

The GPC spin column/ESI-MS methodology with mutated CMV proteases was utilized to characterize the non-covalent binding site of ligand inhibitors [13]. The following illustration demonstrates the use of the GPC spin column screening technique to characterize non-covalent binding of TFMK to specific sites

WY360 TM+2H12+ m/z 317.6 WY854 TM+H11+ m/z 410.3 WY272 TM+H 11+ m/z 453.3

Fig. 2.20 Non-covalent binding competition experiments between IGFr protein target and mixtures of compounds assayed using the GPC spin column/ESI-MS methodology. Mixture 1 compounds are WY360 (MW 633 Da), WY869 (MW 441 Da), and WY-741 (MW 552 Da). Mixture 2 compounds are WY360 (MW 633 Da), WY854 (MW 409 Da), and WY272 (MW 452 Da). Note that WY360 is present in both mixtures so that both mixtures can be correlated. The molecular ion region for the ESI mass spectra are illustrated for: (A) the GPC spin column eluates of incubated components of mixtures 1 and 2 with IGFr protein in a molar ratio (mM) of 56:28, respectively, diluted 2x with water and 10 mL injection; (B) the GPC spin column eluates of incubated components of mixtures 1 and 2 originally each 56 mM per compound, diluted 2x with water and 10 mL injection; and (C) direct infusion of mixtures 1 and 2 with each component 2.2 mM, 1 mL injection. The signal-to-noise ratios for the ESI-MS molecular ion peaks for each of the components of both mixtures are summarized in Table 2.4.

Table 2.4 Relative competitive binding affinities computed from GPC spin column/ESI-MS data of IGFr protein for compounds in mixtures.

Mixture Compound m/z

MS Signal/

MS Signal/

MS Signal/

Normalized

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