MS Binding Assays Quantifying the Bound Marker

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. 

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 [78]). 

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 [80]. 

It had been shown that 50 mM Tris-citrate buffer with 1 M NaCl was a suitable incubation buffer for the binding to GATl [95], The high salt concentration in this incubation buffer caused no problems in the new MS binding assays, since most of the incubation buffer was removed before the liberation step. The separation of the bound from the nonbound marker was conducted by filtration over glass fiber filters as common in radioligand binding assays. 

The results of this saturation assay were validated by direct comparison to conventionally conducted radioligand binding assays using [ H]NO 711 as marker [80, 100]. Not only due to financial considerations, hot/cold dilutions had to be used in the radioligand binding assays, in contrast to the MS binding assays 

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