Inhibitor Screening Using Immobilized Enzyme Reactor Chromatography/Mass Spectrometry
2005-12-01T00:00:00Z (GMT) by
We describe the coupling of capillary-scale monolithic enzyme reactor columns directly to a tandem mass spectrometer for screening of enzyme inhibitors. A two-channel nanoLC system is used to continuously infuse substrate or substrate/inhibitor mixtures through the column, allowing continuous variation of inhibitor concentration by simply altering the ratio of flow from the two pumps. In the absence of inhibitor, infusion of substrate leads to formation of product, and both substrate and product ions can be simultaneously monitored in a quantitative manner by MS/MS. The presence of inhibitor leads to a decrease in product and an increase in substrate concentration in the column eluent. Knowing the product/substrate ratio and the total analyte concentration (P + S), the concentration of product eluting, and hence the relative enzyme activity, can be determined. Both IC<sub>50</sub> and <i>K</i><sub>I</sub> values can then be obtained by direct MS detection of the effect of inhibitors on relative activity. Inhibitor screening is demonstrated using reusable, sol−gel derived, monolithic capillary columns containing adenosine deaminase, directly interfaced to ESI-MS/MS. On-column enzyme activity was assessed by monitoring inosine and adenosine elution. It is shown that the method can be used for automated screening of the effects of compound mixtures on ADA activity and to determine the <i>K</i><sub>I</sub> value of the known inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine, even when the compound is present within a mixture.