MS Binding Assays Quantifying the Nonbound Marker

In radioligand binding assays, binding of the marker is always quantified by the amount of marker bound to the target. The practicality of a procedure that in contrast quantifies the nonbound marker to indirectly determine the amount of bound marker has been shown by applications which examined the binding of fluorescent markers to nicotinic acetylcholine and benzodiazepine receptors [37, 38]. Although very different from conventional binding assays with regard to the design of the binding experiment, the principle to quantify the nonbound marker by LC-MS has also been realized in a sophisticated ''continuous flow'' approach (as described in Chapter 5).

Competition experiments performed as conventional radioligand binding assays are characterized by a nominal marker concentration in the range of its Kd value, while the concentration of the target is set at, by comparison, a significantly lower level (i.e. [Mtot] a Kd » [Ttot]). In radioligand binding assays this set up is possible since the resulting amount of bound marker (TM) is quantified by scintillation counting, that is sensitive enough to reliably measure TM in concentrations « Kd. In MS binding assays where the amount of nonbound marker is to be quantified the situation is completely different. If an MS binding assay of this kind were to be conducted under the same conditions as a radioligand binding assay, the differences between the concentrations of the free marker (DM) that result from changes in the concentrations of the bound marker (TM) would be imperceptible or, at least, extremely hard to detect, because these differences would be so small in relation to the concentration of M that they would hardly exceed the uncertainty regarding the quantitation of M by MS. This problem can be avoided if the concentration of the bound marker (TM) is increased considerably in comparison to the nominal marker concentration (Mtot). This can be achieved by increasing the concentration of the target (Ttot) in comparison to the concentration used in radioligand binding assays. The result of an increasing concentration of the target is exemplified in the following for [Ttot] a Kd while the other conditions remain the same {[Mtot] « Kd, see Eq. (5)}.

Solving Eq. (2) (Section 7.2.1) for the conditions given in Eq. (5) leads to Eq. (6).

Neglecting nonspecific binding, Eq. (6) reveals that a considerable fraction (c. 38%) of the total amount of the marker is bound to the target ([TM] = 0.38 Kd = 0.38 [Ttot] = 0.38 [Mtot]). This means that the changes in the fraction of the bound marker caused in competition experiments, result in a significant change in the concentration of the nonbound marker (M). For saturation and kinetic experiments, however, this concept is more difficult to apply.

In competition experiments that quantify the nonbound marker, as discussed here, the concentration relations are intentionally fixed in a manner that ensures that a significant fraction of the marker is bound ([TM] > 0.1 [Mtot]). Therefore, marker depletion has to be considered when analyzing the data. This can be done by means of Eq. (7), for example [16].

K i: equilibrium dissociation constant of the test compound, IC50: concentration of test compound reducing specific binding of the marker to 50%, [M50]: concentration of the free marker at the IC50-value, [M0]: concentration of the non bound marker in the absence of a competitor, K d: equilibrium dissociation constant of the marker.

Furthermore it has to be taken into account that the nonbound marker has to be quantified out of a matrix containing all the dissolved compounds of the binding sample. To avoid ion suppression of the marker, it is therefore necessary to either use a buffer compatible with MS (i.e. a volatile buffer), or alternatively to remove the matrix of the binding sample prior to quantitation of the nonbound marker.

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