Primary Screening Strategy

The general method for ASMS is shown in Fig. 4.1. In ASMS, the target concentration is generally set at 5-10 mM, so that at equilibrium, ligands with affinities of no weaker than KD @ 10 mM will be significantly bound and, therefore, retained in the ultrafiltration steps. The minimal concentration of each small molecule is dictated by the eventual need to detect ligands by mass spectrometry after several cycles of ultrafiltration and subsequent extraction. In order to ensure detection just above baseline for the vast majority of compounds, which vary in inherent ionization properties and efficiency of mass spectrometric visibility, the starting compound concentration is set at 1.5 mM per compound. The mixture

Fig. 4.1 Schematic of the ASMS experiment format. In primary screening, several thousand compounds are included in a single tube and allowed to equilibrate with the target protein under excess target concentration relative to individual compound ligands. The concentration of each compound is 1.5 mM relative to 5-10 mM target protein. Hence at equilibrium the amount of ligand bound is directly related to both the target concentration and the intrinsic KD of the ligand. Multiple rounds of

Fig. 4.1 Schematic of the ASMS experiment format. In primary screening, several thousand compounds are included in a single tube and allowed to equilibrate with the target protein under excess target concentration relative to individual compound ligands. The concentration of each compound is 1.5 mM relative to 5-10 mM target protein. Hence at equilibrium the amount of ligand bound is directly related to both the target concentration and the intrinsic KD of the ligand. Multiple rounds of

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