Z-fr-amc Cathepsin Muscle

ESI-MS Assay of Cathepsin B

5.2.2.1 MS Assay Development for Cathepsin B

In a first example, we demonstrate the implementation of a homogeneous substrate conversion-based protease assay using the enzyme cathepsin B [17]. Cathepsin B belongs to the group of lysosomal cysteine proteinases, which comprises an important group of enzymes involved in many physiological and pathological processes, such as intracellular protein turnover [18], cancer invasion and metastasis [19, 20]. Cathepsin B catalyzes the hydrolysis of Z-FR-AMC (substrate, N-CBZ-Phe-Arg 7-amino-4-methylcoumarin hydrochloride, Mr 612.3), resulting in two products, Z-FR and AMC (see Fig. 5.3). Figure 5.4a depicts the ESI-MS spectrum obtained after analysis of the enzymatic reaction. It shows the enzymatic cleavage products of Z-FR-AMC, AMC (m/z 175.9) and Z-FR (m/z 456.1). In addition, the spectrum shows the uncleaved substrate (m/z 613.1) and the two SMCs (biotin, m/z 244.9, and cAMP, m/z 329.9). Figure 5.4b shows the ESI-MS spectrum of the reaction mixture after addition of a cathepsin B inhibitor, E-64. The inhibition of cathepsin B results in a strong decrease of the Z-FR and AMC signals. Furthermore, the presence of the E-64 (m/z 358.1) signal demonstrates the potential of the current methodology to simultaneously obtain chemical and biological information of potential enzyme inhibitors.

5.2.2.2 Compatibility of Cathepsin B Assay with MS Detection

A key requirement for the successful on-line coupling of enzyme assays to ESI-MS is the solvent and buffer compatibility. Enzyme assays are mostly performed in nonvolatile buffers, such as HEPES, TRIS, and PBS. Moreover, additives are

Cathepsin Reaction

Fig. 5.4 Monitoring of the enzymatic reaction and cathepsin B inhibition by ESI-MS. MS instrument: Shimadzu LCMS-2010 singlestage quadrupole mass spectrometer. (a) The ESI-MS spectrum obtained after analysis of the enzyme reaction, containing the cleavage products AMC (m/z 175.9) and Z-FR (m/z 456.1); m/z 244.9 and m/z 329.9 belong to the system monitoring compounds, m/z 613.1 belongs to the substrate Z-FR-AMC. (b) The ESI-MS spectrum obtained after addition of the inhibitor E-64 to the enzymatic assay. The signal intensities of AMC (m/z 175.9) and Z-FR (m/z 456.1) are very low as a result of the cathepsin B inhibition; m/z 358.1 corresponds to E-64.

Fig. 5.4 Monitoring of the enzymatic reaction and cathepsin B inhibition by ESI-MS. MS instrument: Shimadzu LCMS-2010 singlestage quadrupole mass spectrometer. (a) The ESI-MS spectrum obtained after analysis of the enzyme reaction, containing the cleavage products AMC (m/z 175.9) and Z-FR (m/z 456.1); m/z 244.9 and m/z 329.9 belong to the system monitoring compounds, m/z 613.1 belongs to the substrate Z-FR-AMC. (b) The ESI-MS spectrum obtained after addition of the inhibitor E-64 to the enzymatic assay. The signal intensities of AMC (m/z 175.9) and Z-FR (m/z 456.1) are very low as a result of the cathepsin B inhibition; m/z 358.1 corresponds to E-64.

added to maintain enzyme activity and stability and to prevent nonspecific surface binding of the proteins. Nonvolatile salts and additives, however, may contaminate the ion source of the mass spectrometer and cause ion suppression in ESI-MS, which results in a decreased MS performance [21]. Consequently, we choose to perform the enzyme-substrate reaction under buffer/salt conditions that are routinely used in ESI-MS, omitting commonly used additives and nonvolatile buffers and salts. Experiments demonstrated that cathepsin B was active in a carrier solution containing solely ammonium formate and 1,4-dithioerythri-

tol (pH 7.0). Organic modifiers, such as methanol or acetonitrile used for elution in (gradient) reversed-phase chromatogram represent another point of concern. In the past, we have shown that biochemical assays can be performed in the presence of organic modifier concentrations up to 15% as long as the reaction time does not exceed 3-5 min. Similar results were obtained for cathepsin B, in which the presence of 10% methanol in the enzyme-substrate reaction (20% in the column) leads to an 11% decrease of product formation. Despite this decrease in enzyme activity, limits of detection (LODs) obtained with the current system compare well with LODs reported, for example, for fluorescence-based readouts for the same enzyme, illustrating the benefits of using ESI-MS as readout technique.

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