Continuousflow Multiprotein Binding Assays Using Electrospray MS

The assay principle shown in Fig. 5.10 has the potential of multiplexing, i.e. performing several assays in parallel, by pumping mixtures of receptors, i.e. streptavidin and anti-digoxigenin and reporter ligands, i.e. fluorescein-biotin and digoxin [29]. Clearly this approach will only be feasible for those assays

Fig. 5.13 On-line continuous-flow, multi-protein biochemical assay. MS instrument: Q-ToF2 (Waters) equipped with a Waters Z-spray electro-spray (ESI) source. Extracted-ion chromatograms of (a) digoxigenin (m/z 391.5), (b) biotin (m/z 245.0), (c) fluorescein-biotin/streptavidin (m/z 390.0) assay and (d) digoxin/anti-digoxigenin (m/z 798.5) assay. Triplicate injections were performed WITH blank (peaks 1-3), 1 mM digoxigenin (peaks 4-6) and 1 mmol L_1 biotin (peaks 7-9).

Fig. 5.13 On-line continuous-flow, multi-protein biochemical assay. MS instrument: Q-ToF2 (Waters) equipped with a Waters Z-spray electro-spray (ESI) source. Extracted-ion chromatograms of (a) digoxigenin (m/z 391.5), (b) biotin (m/z 245.0), (c) fluorescein-biotin/streptavidin (m/z 390.0) assay and (d) digoxin/anti-digoxigenin (m/z 798.5) assay. Triplicate injections were performed WITH blank (peaks 1-3), 1 mM digoxigenin (peaks 4-6) and 1 mmol L_1 biotin (peaks 7-9).

where no cross-reactivity exists between receptors and reporter ligands. Batch experiments (data not shown) revealed that there is no cross-reactivity for ligands binding either to streptavidin or anti-digoxigenin.

The parallel biochemical assay was performed by dissolving both receptor proteins in one solution and the two reporter ligands in one other solution. Both reporter ligands (fluorescein-biotin and digoxin) were pumped together with one pump. The receptors (streptavidin and anti-digoxigenin) were pumped with another pump. Figure 5.13 shows the extracted-ion chromatograms for both reporter molecules. The two lower traces represent the reporter molecule digoxin (m/z 798.5) and the reporter molecule fluorescein-biotin (m/z 390.0), respectively. Triplicate injections of blank, 1 mM digoxigenin, and 1 mM biotin were performed. As expected, the injection of an active compound resulted in an increase in the concentration of the respective unbound reporter molecule. In addition, peaks were observed in the extracted-ion chromatograms of biotin and digoxige-nin. This is a result of the fact that the concentration injected into the carrier solution is a large excess compared to the concentration of receptor present in the carrier solution.

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