Basic Principles

The MS binding assay quantifying the amount of bound marker for the murine GAT1 transporter (mGAT1) was conducted as follows: The source of mGAT1 was a recombinant cell line that expressed the transporter in sufficient density [99]. Of the several possible ligands selective for GAT1, NO 711 (1f in Fig. 7.17) [96, 100] was chosen as a marker because the compound not only shows a high affinity and selectivity for GAT1 but can also be quantified in very low concentrations by LC-ESI-MS/MS (LLOQ = 18 pM; see Fig. 7.14).

It had been shown that 50 mM Tris-citrate buffer with 1 M NaCl was a suitable incubation buffer for the binding to GAT1 [95]. The high salt concentration in this incubation buffer caused no problems in the new MS binding assays, since most of the incubation buffer was removed before the liberation step. The separation of the bound from the nonbound marker was conducted by filtration over glass fiber filters as common in radioligand binding assays.

Fig. 7.14 Example for standard matrix sample. Matrix was obtained by incubation of mGAT1-membrane preparation in Tris-NaCl buffer (@10 mg protein content), subsequent filtration and elution with methanol. This empty matrix was then spiked with NO 711 and [2H10]NO 711 to obtain standard matrix samples for LC-MS method validation purposes. (a) Trace for 0.015 nM NO 711 (mass transition 381 ! 180, m/z). (b) Trace for 1 nM [2H10]NO 711 (391 ! 190, m/z) in the same sample.

For the liberation of the bound marker from the marker-target complex, a simple denaturation with methanol proved to be extremely effective. A drying step before the methanol denaturation and an internal standard (deuterated analog of NO 711, [2H]NO 711; 1g in Fig. 7.17) added to the denaturation reagent further improved the precision of the method. The initially bound marker liberated this way was collected in a 96-well plate and quantified by an isocratic RP-HPLC method followed by ESI-MS/MS detection via the internal standard (see Fig. 7.14). Figure 7.15 gives an overview over the procedure of an MS binding assay with liberation of the bound marker.

7.3.2.1.3 MS Saturation Assays

According to the procedure for the MS binding assays described above, saturation assays with the native marker NO 711 were conducted at mGAT1, after the incubation period was determined (see Section 7.3.2.3). A constant target concentration (10-20 mg protein per well, according to Bradford; @1 nM mGAT1) was incubated with increasing NO 711 concentrations (2-200 nM) and allowed to reach equilibrium. After separation over the 96-well glass fiber filter plates, bound marker was liberated and quantified by LC-ESI-MS/MS. A representative saturation isotherm resulting from these experiments is given in Fig. 7.16.

The results of this saturation assay were validated by direct comparison to conventionally conducted radioligand binding assays using [3H]NO 711 as marker [80, 100]. Not only due to financial considerations, hot/cold dilutions had to be used in the radioligand binding assays, in contrast to the MS binding assays a

Binding Assays

Fig. 7.14 Example for standard matrix sample. Matrix was obtained by incubation of mGAT1-membrane preparation in Tris-NaCl buffer (@10 mg protein content), subsequent filtration and elution with methanol. This empty matrix was then spiked with NO 711 and [2H10]NO 711 to obtain standard matrix samples for LC-MS method validation purposes. (a) Trace for 0.015 nM NO 711 (mass transition 381 ! 180, m/z). (b) Trace for 1 nM [2H10]NO 711 (391 ! 190, m/z) in the same sample.

Fig. 7.15 Schematic flowchart of MS binding assays quantifying bound marker. Incubation of the target (mGATl) in presence of the marker (NO 711) and a test compound is conducted in a 96-well plate. The bound marker is separated from the nonbound marker by vacuum filtration. In the next step the target bound marker remaining on the filter is liberated with methanol. Finally, the liberated marker is quantified by LC-ESI-MS/MS.

Fig. 7.15 Schematic flowchart of MS binding assays quantifying bound marker. Incubation of the target (mGATl) in presence of the marker (NO 711) and a test compound is conducted in a 96-well plate. The bound marker is separated from the nonbound marker by vacuum filtration. In the next step the target bound marker remaining on the filter is liberated with methanol. Finally, the liberated marker is quantified by LC-ESI-MS/MS.

where the entire marker concentration range could be covered with native (''cold'') marker only. As can be seen from Table 7.4, the values of Kd and Bmax determined in both binding studies are in good accord with each other.

Clearly, as indicated by these results, MS binding assays quantifying the originally bound marker can be performed in the same manner as radioligand binding assays to determine the affinity constant of a marker and they are just as efficient.

Saturation Isotherm 711

Fig. 7.16 Saturation isotherm of NO 711 binding to mGATl-membrane fraction as measured in MS binding experiments. One representative example from a series of identical experiments is shown. Total binding of NO 711 (■; 10 mg protein according to Bradford). Nonspecific binding (o) measured as binding of NO 711 in the presence of 10 mM GABA. Each data point depicts the mean G SEM from triplicate values.

Fig. 7.16 Saturation isotherm of NO 711 binding to mGATl-membrane fraction as measured in MS binding experiments. One representative example from a series of identical experiments is shown. Total binding of NO 711 (■; 10 mg protein according to Bradford). Nonspecific binding (o) measured as binding of NO 711 in the presence of 10 mM GABA. Each data point depicts the mean G SEM from triplicate values.

Table 7.4 Comparison of results from saturation binding experiments using native NO 711 in MS binding experiments and [3H]2NO 711 in radioligand binding experiments. All values represent mean + SEM from independent experiments [80].

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