Estimation of Relative Binding Affinities from GPC Spin-Column ESI-MS Data

3 Estimation of Relative Binding Affinities from GPC Spin-Column/ESI-MS Data 

For a variety of ligands in a mixture with the same initial concentration [L], such that [L]g [C] and where the equilibrium concentration of the remaining protein is [P], we can relate back to equilibrium conditions, and using Eq. 7 for computing the ratio of two components subscripted 1 and 2, we obtain  

Note that, in Eq. (8), the concentrations for the complex [C] and related ligand [L] are equal because the ligand is liberated from the complex by denaturing the complex. These non-equilibrium ligand concentration values are obtained by mass spectrometry from the denatured GPC spin column eluate. If the off-rates for the different compounds are the same, k gi = koff2j then  

4 Experimental Determination of the Kd Value from CPC Spin-Column/ ESI-MS Data 

The expression for the equilibrium concentration of the protein-ligand complex [C], described above using Eq. (5), can also be re-written in terms of the total initial protein concentration [Pj such that  

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