Assay Principle

The assay principle for MS-based enzyme inhibition assay is shown in Fig. 5.1. The assay is based on the mass spectrometric detection of reaction products of

Reaction I

Reaction II

Fig. 5.1 Principle of MS-based enzyme assays. Enzyme (E) molecules react with the substrate (S) to form an enzyme-substrate complex (ES), leading, for example, to a subsequent cleavage into two products P1 and P2. P1 and P2 are monitored continuously by ESI-MS. The injection of an inhibitor, I, results in the temporary formation of an inactive enzyme-inhibitor (EI) complex, resulting in a reduction of P1 and P2 and negative peaks in the corresponding mass traces.

Reaction I

Reaction II

Fig. 5.1 Principle of MS-based enzyme assays. Enzyme (E) molecules react with the substrate (S) to form an enzyme-substrate complex (ES), leading, for example, to a subsequent cleavage into two products P1 and P2. P1 and P2 are monitored continuously by ESI-MS. The injection of an inhibitor, I, results in the temporary formation of an inactive enzyme-inhibitor (EI) complex, resulting in a reduction of P1 and P2 and negative peaks in the corresponding mass traces.

Fig. 5.2 Analytical set-up for on-line enzyme assays based on ESI-MS. P1: Carrier/HPLC pump. P2: HPLC pump delivering enzyme solution. P3: HPLC pump delivering substrate solution. 1: Mixing union. 2: Microcoil reactor. In case of on-line coupling to HPLC, the HPLC column is inserted between the autoinjector and the first mixing union. In the first microcoil reactor, the enzyme inhibition takes place (reaction I, Fig. 5.1) whereas in the second microcoil reactor, the enzyme substrate reaction proceeds (reaction II, Fig. 5.1).

Fig. 5.2 Analytical set-up for on-line enzyme assays based on ESI-MS. P1: Carrier/HPLC pump. P2: HPLC pump delivering enzyme solution. P3: HPLC pump delivering substrate solution. 1: Mixing union. 2: Microcoil reactor. In case of on-line coupling to HPLC, the HPLC column is inserted between the autoinjector and the first mixing union. In the first microcoil reactor, the enzyme inhibition takes place (reaction I, Fig. 5.1) whereas in the second microcoil reactor, the enzyme substrate reaction proceeds (reaction II, Fig. 5.1).

the enzyme-substrate reaction at their specific m/z values. The presence of inhibitors in the sample results in a concentration change of both substrate and product due to the temporary inhibition of the enzyme. The hardware implementation is shown in Fig. 5.2. Compounds eluting from the HPLC column or injected into a flow injection (FI) system are mixed with the enzyme solution that is continuously infused via a mixing union. In the first microcoil reactor the reaction between the sample components and the enzyme takes place (reaction I, Fig. 5.1). In the absence of active compounds, the enzyme molecules pass the reactor unaltered and reach the second mixing union where a solution of substrate is infused. In the second microcoil reactor (reaction II, Fig. 5.1), the enzyme substrate reaction is allowed to proceed, the reaction time being determined by the volume of the reactor and the total flow rate which is the sum of the flow rates of the HPLC pump, and the pumps delivering the enzyme and substrate solutions, respectively. Typically the total reaction time is between 30 s and 5 min, depending on the type of enzyme assay. The enzyme substrate reaction results in products that are detected by electrospray ionization MS (ESI-MS) at their respective m/z values. In the absence of inhibitors, a constant concentration of products is formed, leading to a constant baseline of extracted ion current chromatograms (EIC) of the products. An inhibitor that is injected into a FI system or that elutes from the HPLC column temporarily inhibits the enzyme, leading to a decrease of the product concentration that results in a negative peak in the corresponding EICs. In order to correct for ion suppression effects, system monitoring compounds (SMC) are added to both the enzyme and substrate solutions. Only those compounds are considered to be inhibitors that produce a negative peak in the products EICs but no peaks in either of the SMC traces.

Fig. 5.3 Substrate conversion reaction for cathepsin B. Substrate Z-FR-AMC is converted by cathepsin B into two products, Z-FR and AMC, that are employed as reporter molecules at their corresponding m/z traces.

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