MS Binding Assays Quantifying the Nonbound Marker

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 Ka = 0.38 [Tot] = 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]. 

IC50 concentration of test compound reducing specific binding of the marker to 50%, [M50] concentration of the free marker at the ICso-value, [Mo] concentration of the non bound marker in the absence of a competitor, K 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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Fig. 7.5 Schematic flowchart of the competitive MS-binding assay quantifying the nonbound marker employed for dopamine Di receptors. After incubation of the target (Di receptor) in presence of the marker (SCH 23390) and a test compound, the binding samples are centrifuged to separate bound from nonbound marker. The nonbound marker in the resulting supernatant is quantified by LC-ESI-MS/MS without further sample preparation.

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