Application of Maldims in Bioanalysis

Since the end of the 1980s, MALDI has been employed for the analysis of proteins, peptides, oligonucleotides, and polymers in a wide range of applications. Owing to its high tolerance regarding the presence of biological matrices and biological sample constituents, and owing to its advantageous ionization efficiencies for high molecular weight compounds, MALDI-MS has been established as a versatile tool especially in the field of proteomics [2]. Several approaches using MALDI-MS for the monitoring of enzymatic conversions have been introduced over recent years [3-6]. In 2001, Kang et al. developed a high-throughput protocol for the automated determination of enzymatic activities by MALDI-MS [7]. As an enzymatic model system, they used the lipase-catalyzed conversion of rac-1-phenylethylamine (Fig. 8.3).

For reliable quantification, the deuterium-labelled substrate (d5-phenylethyl-amine) was added to the matrix as internal standard. To circumvent the problem of crystal inhomogenities, 100 acceptable spectra were measured from seven to ten different positions of one sample spot and averaged. The MALDI-MS assay was validated with a gas chromatography-based quantification scheme and was found to be in good compliance. This methodology obviously allows a reliable quantification of the low molecular weight analytes of interest. Nevertheless, the need for isotopically labelled compounds as internal standards is still a bottleneck, as these are usually rather expensive or have to be laboriously synthesized.

The potential of the MALDI-MS-based assay scheme for the quantification of low molecular weight products and substrates directly from reaction mixtures has been described by Bungert et al. [8]. The glucose oxidase-based conversion of glucose to gluconolactone and the carboxypeptidase A-mediated cleavage of hippuryl-l-phenylalanine were chosen as model systems (Fig. 8.4).

II III IV

Fig. 8.3 Lipase-catalyzed formation of 2-methoxy-N-[(1R)-1-phenylethyl]-acetamide (III) and (SJ-phenylethylamine (IV). The reaction uses racemic 1-phenylethylamine (I) and ethylmethoxyacetate (II) as educts and is carried out in methyl-tert-butylether. As a byproduct, ethanol is formed.

II III IV

Fig. 8.3 Lipase-catalyzed formation of 2-methoxy-N-[(1R)-1-phenylethyl]-acetamide (III) and (SJ-phenylethylamine (IV). The reaction uses racemic 1-phenylethylamine (I) and ethylmethoxyacetate (II) as educts and is carried out in methyl-tert-butylether. As a byproduct, ethanol is formed.

p-fDJ-glucose (Dj-gluconolactone p-fDJ-glucose (Dj-gluconolactone

Hippuryl Phenylalanine Cleavage
Fig. 8.4 (a) In the presence of oxygen, the glucose oxidase-catalyzed oxidation of b-D-glucose leads to the formation of gluconolactone. (b) Carboxypeptidase A selectively cleaves the substrate, hippuryl-L-phenylalanine, thus leading to the formation of hippuric acid and phenylalanine.

Time-resolved reaction profiles for both enzymatic reactions were obtained by simultaneous determination of the respective substrate and product concentrations without the need for time-consuming sample preparation steps. The results were in good agreement with those from a standard UV absorbance-based assay. In another study by the same group, a liquid ionic matrix was employed instead of using a crystalline solid matrix, thus minimizing the negative effects on the quantification by sample spot inhomogenities [9]. The method was applied to screen the enzymatic activity of ten pyranose oxidase variants towards glucose (Fig. 8.5).

Each sample was mixed with the ionic liquid matrix (2,5-dihydroxybenzoic acid/pyridine) containing 13C-labelled glucose as internal standard and spotted on the target. MALDI-MS analysis generated reaction profiles by the simultaneous determination of product and substrate concentrations for each enzyme variant. The reaction profiles could be used to sort the enzyme variants into five different classes.

In 2006, Greis and co-workers reported on the application of MALDI-TOF MS as a tool for rapid inhibitor screening [10]. Different kinases (protein kinase C-a, cAMP-dependent protein kinase) in combination with their substrates were assayed, and the inhibitory potencies of staurosporine and three novel compounds were determined. For all four compounds, IC50 values could be determined, and

p-fDJ-glucose (DJ-glucosone

Fig. 8.5 The pyranose oxidase-catalyzed oxidation of b-D-glucose leads to the formation of glucosone. Educt and product differ by 2 Da.

p-fDJ-glucose (DJ-glucosone

Fig. 8.5 The pyranose oxidase-catalyzed oxidation of b-D-glucose leads to the formation of glucosone. Educt and product differ by 2 Da.

staurosporine was found to possess the highest inhibitory potency. In the field of drug discovery, selectivity of an inhibitor, i.e. of a potential drug substance, plays a significant role. As many enzymes exist as members of enzyme families, an inhibitor would only be useful as a drug candidate in those cases, where it selectively inhibits the target enzyme. Therefore, the inhibitory potency of staurosporine towards both protein kinases was assayed by means of MALDI-TOF MS. In accordance with literature data, it was found that stauposporine shows ten times higher inhibitory activity for protein kinase C-a than for cAMP-dependent protein kinase. Although LC/MS-based approaches are still the method of choice in the field of quantitative enzyme activity screening, MALDI-TOF MS has thus shown to be a versatile alternative in all those cases where minimal sample preparation is required and high-throughput analysis is desirable.

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