Summary and Perspectives

As the examples described here show, the goals that so far have been pursued with radioligand binding assays can, in principle, also be achieved with MS binding assays based on the mass spectrometric quantitation of native, i.e. nonlabeled markers. MS binding assays can be conducted in an experimental setup in analogy to radioligand binding assays, i.e. quantifying the amount of marker bound to the target. In contrast to radioligand binding assays, however, the quantitation in MS binding assays does not proceed at the level of the target-marker complex directly after its separation from the binding sample. In MS binding assays, the marker is liberated from the target-marker complex, before it is quantified by LC-ESI-MS/MS. In this way, the basic types of binding assays, saturation, competition and kinetic assays can be realized. Under certain conditions (with a significant amount of Mtot being bound to the target T), MS binding assays can also be arranged in a way that the nonbound marker can be quantified by LC-ESI-MS/MS directly from the supernatant obtained by centrifugation of the binding sample.

Irrespective of certain limitations, this method offers the opportunity to efficiently conduct competitive binding assays.

Although the applications presented here are exclusively based on membrane-bound targets, MS binding assays are not restricted to them. Generally, every kind of target can be examined in MS binding assays as long as suitable markers are available. The search for suitable markers, however, is in contrast to radioligand binding assays (or assays based on fluorescent markers), greatly facilitated by a much wider repertory of potential markers, since they are used in their native, i.e. unlabeled form. The most demanding task in MS binding assays, the reliable mass spectrometric quantitation of the marker, is increasingly facilitated by the continuously improving sensitivity of modern mass spectrometers. If the sensitivity of the mass spectrometer tends to limit the quantitation of the marker in the binding assay, it is still possible to partly compensate this problem by choosing a higher target concentration than commonly used in radioligand binding assays.

The throughput that can be achieved in MS binding assays depends on both the workflow chosen for the binding experiments as well as the mass spectromet-ric quantitation of the marker. In the binding experiments itself, the throughput is - just as in radioligand binding assays - primarily dependent on the separation step. MS binding assays based on filtration as separation step can be performed in a 96-well plate format just as easily as radioligand binding assays. But in the analytical setup described here, HPLC dictates the speed of quantitation. Although quantitation in MS-binding assays generally requires more time than measuring radioactivity, the applications above show that it is possible to process several hundred samples a day, even with a very simple instrumentation. Since high-sensitivity quantitation of an analyte in a biological matrix by LC-MS is a quite frequent topic in the life sciences (e.g. in pharmacokinetics, see Chapter 13) there are a number of possibilities to significantly accelerate this process [78, 102-104].

Even though MS binding assays follow the principle of radioligand binding assays, their potential significantly exceeds that of the radioligand binding assays as shown by the applications described above. It is, for example, possible to use the marker in the binding assays, even in very high concentrations, or to identify structurally unknown hits in a library. To mention only one further example for other feasible options, it should be possible to track several targets simultaneously in one MS binding assay.

In summary, MS binding assays can be applied comparatively easily and universally without the inherent disadvantages of labeling. Thereby their reliability is approximately equal to that of radioligand binding assays. Therefore, it can be expected that MS binding assays will find increasing use in drug discovery.

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