B Esi Mo mz 304

D0200339 12 (0.435) Sm (SG, 2x5.00); Cm (7:54-1:4x4.000) 1 : TOF MS ES-

D0200339 12 (0.435) Sm (SG, 2x5.00); Cm (7:54-1:4x4.000) 1 : TOF MS ES-

D0200344 13 (0.446) Sm (SG, 2x5.00); Cm (7:55-1:4x4.000) 1 : TOF MS ES-

Fig. 2.7 ESI mass spectra of ligands present in GPC spin column eluates have a linear response with increasing concentration: ESI (positive ionization mode) mass spectral analysis of the eluate from the GPC spin column titration of WY252 (MW 457 Da) with MMP-1 where the molar ratios of MMP-1/ WY252 are constant at 1:5 while their individual concentrations linearly increase. The volume injected for each sample was

30 |mL. (A) MMP-1 alone at 50 |mM and (F) WY252 alone at 250 |M, respectively. (B-E) increasing amount of MMP-1: (B) 20 |M, (C) 30 |M, (D) 40 |M and (E) 50 |M; and increasing amount of WY252: (B) 100 |M, (C) 150 |M, (D) 200 |M and (E) 250 |M. Same absolute intensity scale for all panels. Reprinted from reference [15] with permission from the American Chemical Society

Fig. 2.7 ESI mass spectra of ligands present in GPC spin column eluates have a linear response with increasing concentration: ESI (positive ionization mode) mass spectral analysis of the eluate from the GPC spin column titration of WY252 (MW 457 Da) with MMP-1 where the molar ratios of MMP-1/ WY252 are constant at 1:5 while their individual concentrations linearly increase. The volume injected for each sample was

30 |mL. (A) MMP-1 alone at 50 |mM and (F) WY252 alone at 250 |M, respectively. (B-E) increasing amount of MMP-1: (B) 20 |M, (C) 30 |M, (D) 40 |M and (E) 50 |M; and increasing amount of WY252: (B) 100 |M, (C) 150 |M, (D) 200 |M and (E) 250 |M. Same absolute intensity scale for all panels. Reprinted from reference [15] with permission from the American Chemical Society molar excess of drug. Similarly for the study of weak binders, protein concentrations 15-200 |M with 1-100 times molar excess drug can be used.

The unique property of the GPC spin column experiment is that the volume of sample loaded on to the spin column is the same volume of the eluate obtained after gentle centrifugation. Furthermore, when two GPC spin column experiments are performed, as described above, initially with an incubated protein-ligand sample in one column and with a ligand sample without protein as a control in another column, each followed by repeated elutions with buffer solutions, the most significant ligand concentration difference between the protein-ligand sample and the control sample is between the first collected fractions. This is illustrated in Fig. 2.5 for GPC spin column eluate fractions for a protein kinase A (PKA)-staurosporine sample and a control sample of staurosporine without PKA [16]. These fractionation experiments verify the fundamental principle of the GPC spin column methodology that only the first fractions of the protein-ligand mixture and the control experiments are needed for non-covalent binding studies for drug screening.

Dose response titrations were used to demonstrate that the non-covalent binding in the GPC spin column/ESI-MS assay is a function of the protein and ligand concentrations. Methyl orange is a very weak binder to bovine serum albumin (BSA) with a Kd of 450 mM and its passage through the GPC spin column can be visually monitored. A variety of equimolar concentrations of BSA and methyl orange were analyzed using the GPC spin column/ESI-MS methodology, as illustrated in Fig. 2.6 for both the positive and negative ESI ionization modes. The chemical noise level in these experiments is about 45 counts (note: the ion counts are indicated in the upper right hand corner of each spectrum.) Only background traces of the molecular ions for methyl orange (m/z [M+H]1+: 306; [M-H]1-: 304) were observed in the mass spectra for equimolar concentrations 12.5 mM and 25 mM, i.e., the ion abundances were slightly greater than the chemical noise, while the methyl orange response grew with increasing equimolar concentrations from 50 mM to 200 mM. Visual observations of the column confirm these results. At the two lower concentrations, the orange color of methyl orange was confined to the top of the GPC spin column and with increasing concentrations the orange color moved down the column towards the top of the spin column frit, consistent with the ESI-MS intensity observations. Similar dose response titration studies have been reported with stronger binders, e.g., matrix metalloproteinase-1 (MMP-1) protein with a substituted hydroxyamide WY252 with an IC50 of 9.9 mM (see Fig. 2.7) [15].

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