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Fig. 6.5 A diagram of the modified staircase approach. In this procedure, ligand and void marker are infused at increasingly higher concentrations. It differs from the normal procedure in that column washing between infusions is not performed. This is the simplest of FAC techniques for measuring ligand Kd.

Fig. 6.5 A diagram of the modified staircase approach. In this procedure, ligand and void marker are infused at increasingly higher concentrations. It differs from the normal procedure in that column washing between infusions is not performed. This is the simplest of FAC techniques for measuring ligand Kd.

where:

A plot of [A]0 + y versus reciprocal breakthrough volume supports the determination of Bt and Kd by linear regression analysis. As with all FAC methods for ligand characterization, the cartridge does not require precalibration before a measurement is made, because Bt is a product of the measurement. One disadvantage of this method is that any error in the earlier measurements is carried forward in subsequent ones; however the speed of these measurements and the ability to accurately measure V0 usually makes this the method of choice.

These sorts of analyses are limited only by the ability of the chosen mass spectrometer to detect the test-compound under infusion-buffer conditions. In many cases, there will not be a problem even at low nanomolar compound concentrations, but in others MS will be hard-pressed to detect compounds even at micromolar concentrations. The online FAC-MS system will always be challenged by the inherent incompatibility of routine assay buffers with MS, but there are opportunities to reconfigure either the ion source or the buffer composition for the demands of the analysis. For example, detection of steroids may benefit from an APCI source over electrospray, whereas a MALDI source may have greater tolerance for higher ionic strength buffers. A powerful alternative involves the insertion of a FAIMS (high field asymmetric waveform ion mobility spectrometry) device [17] after electrospray ionization. We have shown that compounds buffered in full-strength PBS (@150 mM NaCl) can be successfully detected in an online FAC-MS experiment, whereas a simple selected ion reaction monitoring of the compound was unsuccessful (data not shown). In our experience, sub-micromolar to low millimolar Kd values can be successfully characterized with the on-line method under a wide range of buffer conditions. Every increment in MS sensitivity further extends the Kd range over which the direct method can function.

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