Application of MALDI-MS in Bioanalysis

For reliable quantification, the deuterium-labelled substrate (ds-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). 

Each sample was mixed with the ionic liquid matrix (2,5-dihydroxybenzoic acid/pyridine) containing C-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 

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