Step 1 of our qµD process is the determination of the appropriate concentration of the small molecules needed for detection. Non-fluorescent or fluorescently labelled compounds can be analysed by RP-HPLC. For non-labelled compounds, a concentration series of 10, 25, 50, 75 and 100 μM is recommended, with 25–50 μM usually falling in the middle of the linear range. Our lab used this method to confirm hit compounds arising from on-bead screening [
12]. Standard library compounds or fragments that do not contain aromatic components cannot be detected by RP-HPLC in sufficiently low concentrations for the qµD process. Instead, these compounds were analysed by LC–MS. To find the appropriate concentration in this case, it must first be established whether the compounds ionise at all, and in which detection mode. The LC–MS method for concentration determination was detailed in
Section 3. This protocol was established for analysis by a Thermo Fisher
TM LTQ Orbitrap XL
TM. Adaptation of the protocol to other mass spectrometry instruments should be straightforward for any lab proficient in mass spectrometry. In
Scheme 1 (Step 1), the concentration range comprising of 50, 100, 500, 1000 and 1500 nM, with 250–500 nM being in the middle of the linear range for most compounds, was derived from an Orbitrap instrument. Step 2 of the qµD analysis process involves simulating the dialysis process. This will inform the experimentalist as to the most suitable concentration of the target protein, to maximise the disparity of compound concentrations between the two chambers, within the detection limits. Therefore, this step is critical to maximise the accuracy of the method. Step 2 was described in more detail in
Section 2.2. Step 3 involves testing whether the compound equilibrates uniformly enough between the two dialysis chambers in the absence of the protein, to make it suitable for incubation with the target protein and one or more negative control proteins in Step 4, to test the compounds potential for unselective or nonspecific binding. It is well known in biochemistry that proteins need to be tested for their folding state and aggregation potential. On the other hand, compounds contained in screening libraries, and—more importantly—primary screening hits, are not as routinely investigated for inherent problematic behaviours. These behaviours often lead to false positive and false negative results. Dialysis offers a versatile way to address this; it can be used to test surface binding as a function of buffer composition, pH, additives and other assay variables, as well as to identify dynamic aggregation and promiscuous binding as a function of concentration. Using the predefined compound concentration,
pc is determined. This should ideally be below 1.10 after an appropriate time period in the target incubation step. Now, having simulated the concentration of the target protein, and proven the purity and suitability of the test compound, Step 4 of the qµD process may be performed. In
Scheme 1 (Step 4), a single incubation experiment with the target protein and one with BSA as a control is shown. Additional selectivity and specificity controls can be integrated as needed. The time frame for equilibration can vary as a function of the concentration of the compounds, affinity to the target or temperature, however a study by Walters [
13] demonstrates equilibrium of 18 diverse drugs covering a wide range of physicochemical properties is achieved within 6 h. Similar studies by van Liempd et al. [
14] and Ye et al. [
15] further demonstrate 6 h to be an adequate equilibration time. We started with 6 h of incubation, defined by the compound-only experiment. To determine the partition coefficients, the compound concentration needs to be analysed in both compartments. A high concentration of protein can interfere with the chromatographic separation, making it desirable to remove the protein prior to analysis. To this end, methods for protein denaturation and precipitation using organic solvents, acid or heat have been developed [
13]. However, we have found that coprecipitation of the compound with the target protein may occur to a certain extent, in line with observations by Vanholder et al. [
16], reducing the reliability of the subsequent measurement. Furthermore, adding the denaturation agent to the sample results in a large volume increase, which need to be offset and the original volume established to perform a quantitative analysis. Hence, we present here a method where no protein precipitation or chromatography of the protein containing samples is needed to analyse samples by RP-HPLC. In addition, for the MS analysis, only a simple dilution is required (due to the sensitivity of the mass spectrometer used), which does not compromise the sample quality. At this dilution it was found that the amount of protein present was compatible with our LC–MS setup and therefore analyses could occur without interfering with the compound detection.