Application of SAMDI in Bioanalysis

The use of functionalized monolayers in monitoring enzymatic activities was tested using b-1,4-galactosyltransferase as model enzyme and immobilized N-acetylglucosamine as substrate. The enzyme solution was incubated on SAM-modified target surfaces. Subsequent to incubation, the target was rinsed, an appropriate matrix was applied, and the surface was analyzed by means of MALDI-MS. By varying the incubation times, time-resolved reaction profiles were obtained. The yield of the enzymatic conversion could be calculated from the ratio of the product signal to the sum of product and substrate signal intensities [22].

An approach to multiplexing analysis was presented by Min et al. [23], who de-velopped a SAMDI-based assay scheme to screen for the activity of different kin-ases. In this assay scheme, peptide substrates were used that are specific for one type of kinase. A mixture of four substrates was immobilized on the SAM. After incubation with an appropriate kinase, the target surface was rinsed, thus stopping the reaction. Matrix was deposited on the surface and MALDI-MS analysis was carried out (Fig. 8.13). By monitoring the signal intensities for the substrates

Fig. 8.13 A multiplexing SAMDI-MS assay. In this case, a mixture of three substrates is immobilized on the SAM. After incubation with appropriate enzyme solutions, the enzymatic reaction is quenched by rinsing the surface. Subsequently, matrix is deposited on the surface and MALDI-MS is carried out. Consumption of all three substrates can thus be determined in parallel.

Fig. 8.13 A multiplexing SAMDI-MS assay. In this case, a mixture of three substrates is immobilized on the SAM. After incubation with appropriate enzyme solutions, the enzymatic reaction is quenched by rinsing the surface. Subsequently, matrix is deposited on the surface and MALDI-MS is carried out. Consumption of all three substrates can thus be determined in parallel.

and the products, enzymatic activity was determined, thus demonstrating the feasibility of the SAMDI approach for multiplexing analysis of enzymatic activities.

In a second series of experiments, the quantification of enzyme inhibition by means of SAMDI-MS was studied. For quantitative studies, two different kinases were incubated separately with varying amounts of known inhibitors on the modified target surface. The inhibition of the respective reactions in the presence of the inhibitors was determined by MALDI-MS, and IC50 values for both kinases could be generated. However, the reported IC50 value for the inhibition of casein kinase I was significantly higher than the value that had been determined from a different assay scheme. A possible explanation for this may be the differences between a liquid-phase reaction and a surface/liquid-phase reaction. Furthermore, the general problem of accurate quantification of the analytes in MALDI may have been contributed to this deviation.

Furthermore, SAMDI-MS methods have been developed that may be used as screening procedure for the identification of anthrax lethal factor inhibitors [24]. Anthrax lethal factor is a zinc-dependent protease. An oligopeptide, which is cleaved by the enzyme at a proline position, served as a model substrate and was immobilized on the SAM-modified surface of a MALDI target. The target plate comprised an array of 100 gold-coated and SAM-modified sample spots. For initial screening, different mixtures - each consisting of eight potential inhibitors -were added to an aliquot of the enzyme solution. These mixtures were then incubated on the target sample spots. After the enzyme reactions had been stopped, matrix was added and MALDI-MS analysis was performed. Inhibition activity was assessed, when no or only small product signal intensity was observed. Then, the compounds present in those wells, where complete or partial inhibition was observed, were screened individually. Thus, one compound could be identified which completely inhibited the enzymatic activity of anthrax lethal factor. Incubation of the enzyme in the presence of varying amounts of inhibitor and determination of the relative amount of product formed during the reactions enabled quantification of the inhibition activity. Based on these assays, an IC50 value for the inhibitor could be calculated that fitted well to the value obtained by a photometric reference assay. With this example, the high potential of SAMDI-MS for chemical screening of inhibitory activity in a high-throughput environment such as drug discovery has been impressively demonstrated. A variation of the SAMDI-MS approach for monitoring enzymatic conversions has been presented by Min et al. within a so-called pull-down assay scheme (Fig. 8.14) [25].

In this pull-down assay, the enzymatic reaction is carried out completely in solution. Samples taken from the reaction mixture are then transferred to a SAM-modified MALDI target, on which the remaining substrate and the reaction product are selectively immobilized. Subsequent to the extraction of the analytes, the target is rinsed, treated with matrix, and MALDI-MS analysis is carried out. A major advantage of this assay scheme is that the inherent danger of negative influences on the reaction kinetics, which may be caused by immobilization of the substrate as in standard SAMDI-MS-based assay formats, is circumvented. Additionally, by selective extraction of the analytes of interest and removal of the other

Fig. 8.14 Scheme of a pull-down assay. The enzymatic reaction is completely carried out in solution. Upon enzyme addition, substrate is consumed, and product is formed. Sample aliquots are taken at several time points from the reaction mixture and are taken to a SAM, which has been modified with selective end groups. The latter are able to bind both substrate and product. Finally, matrix is added, and the SAM is analyzed by means of MALDI-MS.

Fig. 8.14 Scheme of a pull-down assay. The enzymatic reaction is completely carried out in solution. Upon enzyme addition, substrate is consumed, and product is formed. Sample aliquots are taken at several time points from the reaction mixture and are taken to a SAM, which has been modified with selective end groups. The latter are able to bind both substrate and product. Finally, matrix is added, and the SAM is analyzed by means of MALDI-MS.

reaction mixture constituents, the chemical background during the MS analysis can be significantly reduced.

Recently, a SAMDI-MS assay was described by means of which endogenous caspase protease activities in cell lysates can be determined [26]. Similar to the assay used to determine anthrax lethal factor inhibitors, peptide substrate SAMs for either caspase-3 or -8 were treated with cell lysates. In contrast to fluorescence assays, also longer peptide substrates could be used, thus enabling a better resolution of the two caspase activities.

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