Another promising setup for MS binding assays is the quantitation of the bound native marker. Such MS binding experiments would allow ''transferring'' already established radioligand binding studies with relatively small effort to the format of MS binding assays and produce comparable results. Additionally, this approach has several other advantages.
Considering the higher target concentrations generally necessary for MS binding assays with quantitation of the nonbound marker, it is obvious that a depletion of the test compound can very easily occur (particularly if Ki < Kd). In case of MS binding assays with quantitation of the bound marker this should be less of a concern, as the target concentrations are, in general, distinctly lower (see Section 7.3.1 and ).
It is also interesting to note that MS binding assays performed in analogy to a radioligand binding assay determining the amount of bound native marker allows binding assays that would only be possible with radioligands in some kind of a ''mixed mode'': it is not always possible to examine the entire concentration range with pure radioligands (''hot only'') due to the restricted availability, high costs or low affinity of the radioligand. Therefore, conducting saturation assays requires that mixtures of labeled and unlabeled, i.e. ''hot'' and ''cold'', markers are used [36, 68].
To conduct MS binding assays with ligand liberation, similar prerequisites have to be met as generally described for MS binding assays (see Section 7.3). Besides high affinity and selectivity of the marker for the target, these include in this setup a quantitation method with a sufficient sensitivity, or in other words with a lower limit of quantitation (LLOQ) low enough to reliably quantify the low quantities of marker commonly present in such studies. For this last point, one important aspect is also the target density of the target material used in the assay: A sufficient amount of marker should be bound to ensure that a satisfactory signal intensity is reached. Yet binding of the marker and ligand should still be kept low enough to ensure that depletion is less than 10%. Also in this context, cell lines heterologously expressing the desired target seem advantageous [69-71].
The separation of the target-marker complex from the free marker can be achieved with different techniques (e.g. centrifugation or filtration). When conducting binding experiments with membrane-bound targets, filtration is generally preferred due to its speed and effectiveness.
One of the essential steps when developing MS binding assays quantifying the bound marker is the liberation of the marker from the target-marker complex retained, e.g. in the filter, and its quantitation in the relevant eluate [39, 72-75]. A very practical way to implement this is using filter plates in the 96-well format with suitable vacuum manifolds where elution can be directed to another 96-well plate. Generally, glass fiber filters are often used in radioligand binding assays which are also available in different versions in the 96-well format .
The liberation of the marker from the target-marker complex after separation should be complete and reproducible and include both the specifically and non-specifically bound marker. To achieve this, common methods for protein denatu-ration should be suitable (e.g. change in pH value, addition of organic solvents, chaotropic salts, detergents or increase in temperature) [76, 77]. However, it has to be kept in mind that the denaturation method should not interfere with the subsequent quantitation. When using ESI-MS for quantitation for example, high salt concentrations can lead to signal suppression and impair the LLOQ of the method [78, 79]. Therefore, denaturation with organic solvents seems to be more advantageous when using ESI-MS for quantitation.
A practical advantage of MS binding assays quantifying the bound marker after liberation is the "decoupling" of biological binding assay and analytical MS quantitation. In this case the choice of buffer is less restricted as the incubation buffer is mostly removed in the separation step and therefore can contain, for example, high salt concentrations or other additives that would negatively influence MS quantitation. For the denaturation the only aspect that has to be kept in mind is that a method is chosen with which the marker is transferred into a solution that does not interfere with the quantitation.
However, the solution obtained after denaturation might include, depending on the application, other components besides the liberated marker ("matrix"). If a small amount of target material is used in the binding assay, the quantity of remaining matrix will be so low that it hardly disturbs the quantitation and the sample can be measured directly by LC-MS without further sample preparation (e.g. membrane filtration or solid phase extraction ).
In the following, an example of this new kind of MS binding experiment is presented as a straightforward alternative to conventional radioligand binding assays and suitable for the performance of saturation, competition and kinetic binding assays .
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