Assay Principle

Next to the detection of enzyme inhibition, ESI-MS can also be used to monitor protein-ligand interaction, employing an assay format similar to fluorescence-based receptor assays. Using a similar continuous-flow analytical screening system as shown in Fig. 5.2, a competitive assay can be set up using ESI-MS to measure the interaction of the analyte(s) with an affinity protein such as an antibody, receptor or enzyme [28]. Figure 5.10 shows the equilibrium reactions that form the basis of the assay concept. In a first step, the sample was injected into a con-

Fig. 5.10 Principle of competitive ligand binding MS assays. Protein (P) molecules react with the test ligand (L) to form a protein-ligand complex (PL). The extent of complex forming is monitored by the addition of a bioactive reporter ligand (R) resulting in the formation of protein-reporter complex (PR). The concentration free R is directly dependent on the concentration and affinity of L; R is monitored by ESI-MS at its corresponding m/z trace.

Fig. 5.10 Principle of competitive ligand binding MS assays. Protein (P) molecules react with the test ligand (L) to form a protein-ligand complex (PL). The extent of complex forming is monitored by the addition of a bioactive reporter ligand (R) resulting in the formation of protein-reporter complex (PR). The concentration free R is directly dependent on the concentration and affinity of L; R is monitored by ESI-MS at its corresponding m/z trace.

tinuous-flow reaction system and allowed to react with the affinity protein for 1020 s. In the second step, a reporter ligand was added to saturate the remaining free binding sites of the affinity protein. The reaction time was 10-20 s and depended mainly on the binding constant of reporter ligand-affinity protein complex. The reaction time was chosen in a way that the association of free affinity protein molecules with the reporter ligand is favored whereas the dissociation of the analyte-affinity protein complex is negligible. Finally, the concentration of free reporter ligand was detected using ESI-MS in the SIM mode.

Generally, in biochemical analysis a phosphate buffer is used to mimic physiological conditions (about pH 7.5). The percentage of organic modifier is usually kept as low as possible to prevent denaturation of the proteins. In addition, a blocking reagent such as Tween-20 is added to prevent non-specific binding of the protein (and protein-ligand complex) to the surface of reaction capillaries. However, nonvolatile additives in the eluent, such as phosphate buffer and blocking reagent, are not compatible with MS detection. Various reaction conditions were monitored using a series of MS-compatible solvents and compared with the responses observed in the fluorescence detection.

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