The increasing performance of mass spectrometry as well as the development of particularly gentle but also effective ionization techniques like matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) provided the opportunity to directly study binding of native ligands at defined targets [51, 52] (see also Chapter 1). The first MS based methods to measure target-ligand interactions were described in the beginning of the 1990s . Later on, in 1995, Henion and coworkers reported the determination of the affinity constants of gly-copeptide antibiotics like vancomycin at peptides representing target partial structures. They attained this goal by analyzing the target-ligand complexes and the free ligands in parallel directly in the gas phase by ESI-MS . By now, a great variety of different methods to study target-ligand interactions qualitatively or quantitatively based on mass spectrometry have been published [50, 52, 55, 56].
In contrast to most of the other detection methods that can be employed for the characterization of target-ligand interactions, mass spectrometry also allows the identification of structurally unknown ligands. Due to this almost exclusive potential, mass spectrometry has an outstanding position in the screening of combinatorial libraries. The principle to filter hits based on their affinity to the target from a set of different test compounds is called affinity selection. The majority of screening techniques based on mass spectrometry follow the affinity selection principle, and several of these have achieved an impressive efficiency, for example the automated ligand identification system (ALIS, see Chapter 3) established by NeoGenesis (now part of Schering Plough) and affinity selection mass spectrometry (ASMS, see Chapter 4) established by Abott or SpeedScreen from Novartis .
Although these and many other successful approaches for the MS determination of a compound's affinity are regarded as being well established, their application is still reserved to specialists as they require the application of comparatively complex and high sophisticated techniques. The method presented below is therefore deliberately employing a comparatively simple principle: the MS quan-titation of native markers in binding assays that are conducted in analogy to ra-dioligand binding assays. This method has been termed ''MS binding assays''.
MS binding assays share all the advantages of conventional radioligand binding assays, the principle is very simple, they are robust, comparatively cheap, flexible and universally applicable, without the disadvantages caused by a label. Basically, the binding assay itself can be performed exactly as a radioligand binding assay. However, after separation of the bound from the nonbound native marker, the marker quantitation is done by mass spectrometry. As the quantitation method for the marker can be used for all binding samples in the same way, MS analysis creates a modicum of effort. The prerequisites for the native marker are first of all - just as in radioligand binding assays - that it shows a high affinity and selectivity for the target and, at the same time, as little nonspecific binding as possible. Secondly, the marker should be quantifiable by mass spectrometry with a sensitivity as high as possible.
In one point, however, MS binding assays differ fundamentally from radioligand binding assays. In radioligand binding assays it is of little importance whether the marker (i.e. the radioligand) is free or bound to the target during quantitation, since measuring the radioactivity detects both the bound and the already dissociated marker in the same way. This is not the case in MS binding assays, where the free marker and the bound marker (i.e. the target-marker complex) give rise to different signals. Additionally, the exact mass spectrometric quantitation of the marker poses a formidable challenge, particularly if the concentrations to be determined lie in the picomolar range or below (as typical in radioligand binding experiments).
As it is often difficult to prevent dissociation of the target-marker complex during quantitation, this problem can be solved by first separating the target-marker complex from the nonbound marker and, in a second step, liberating the bound marker from the complex for the mass spectrometric quantitation. MS binding studies of this type will be discussed in Section 7.3.2. Alternatively, MS binding studies can be also conducted by quantifying the amount of nonbound marker in the binding sample instead of bound marker (as the amount of bound marker can be calculated from the amounts of total marker and nonbound marker). While the latter method allows to complete the experiment without the additional step of liberating the marker from the target-marker complex it is subject to some restrictions concerning the experimental procedure. Examples for this procedure are given below in Section 7.3.1.
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