Competition Assays for mGATl

For the establishment of saturation and competitive MS binding assays described above, association and dissociation assays with mass spectrometric quantitation of the native marker had also been conducted. 

In association assays, a constant NO 711 concentration (in the region of fCj) was incubated with the target. Using the previously described method, the binding experiments are terminated after different periods of time and quantified by LC-ESI-MS/MS. The binding curve derived from the data is shown in Fig. 7.20. It 

A further characteristic of ligand-target interaction is the dissociation rate constant. Dissociation experiments are not only an important criterion for the establishment of binding assays, they also can show the reversibility of the specific marker binding at the target [16], which is usually verified by dissociation experiments (see also Section 7.2.1). In the MS binding assays presented here, GABA was used as a competitor to initiate dissociation. The experiment was then termi- 

Knowing the rate constants of the target-marker binding experiment gives a different way to determine the dissociation constant independently from saturation experiments [see Eq. (4) in Section 7.2.1]. For this purpose, the association rate constant fe+i has to be calculated from feobs according to Eq. (8)  

This yielded a fe+i of 0.0091 + 0.002 nM mm for NO 711 binding to mGATl. Hence, the equilibrium dissociation constant calculated from kinetic MS binding experiments resulted in = 11.7 2.5 nM. This is in good accord with Kj determined in MS saturation binding experiments and confirms the validity of the new setup. 

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