In the retesting and deconvolution phase of the procedure new compound mixtures were made based on the results of primary screening. These contained from nine to 14 compounds and no monoisotopic mass redundancy. Since most mass spectrometric peaks picked as hits in the primary screen contain more than one compound, and only one compound per peak is likely to be a binder, the non-mass redundant retest mixtures are unlikely to contain more than a few bona fide ligands, so once again target excess is maintained. Both the initial (round zero, R0, prior to first round of filtration) and final (round three, R3, after three rounds of selection) mixtures are sampled. The free target concentration is in excess over individual ligands, so the amount of compound bound at equilibrium can be estimated according to Eq. (1).
The amount of free ligand is disregarded in this estimation. KD can be estimated according to the following, where R0 and R3 represent either a raw signal intensity or signal to local background ratio according to Eqs. (2-4).
(average per round)
During the deconvolution phase of screening, careful control of the pre- and post-filtration volumes are required to ensure both a rank ordering of binding strengths and estimation of the KD value. The post-filtration volumes are controlled by use of a novel pressure limited equilibrium filtration device for selection steps. Operationally, all of R3 is sampled for analysis and 10% of R0 is sampled. Therefore, when the compound KD is equal to the protein concentration, the R3 signal will be approximately equal to the R0 signal, and an R3 signal generated through selection in the absence of protein will be 100-fold (rather than
1000-fold) less than the R0 signal. Compounds whose KD values are ten-fold or more below the protein concentration appear as peaks with approximately ten times the intensity of the R0 peak, assuming a linear dose response in the mass spectrometer. Using the example of a compound with KD = [protein] and a starting compound concentration of 1.5 mM in a volume of 400 mL, 75 pmol of compound remains after three rounds (600 pmol divided by two, three times). Since the final samples are split into three aliquots, for positive ion analysis, negative ion analysis, and a backup sample as needed, approximately 25 pmol of material is available for analysis. By measuring the signal intensity or signal/background ratio for a compound before and after selection, the KD value can be estimated.
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