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Fig. 5.17 Demonstration of MS-based bioassay functionality using a plant extract. MS instrument: Ion-trap mass spectrometer (LCQ Deca, Thermo Electron). (a) MS analysis of pure extract by direct injection onto restricted-access column 2 in the absence of affinity protein. (b) Analysis of the same natural extract spiked with digoxin using the labelfree MS assay method as shown in Fig. 5.15.

Fig. 5.17 Demonstration of MS-based bioassay functionality using a plant extract. MS instrument: Ion-trap mass spectrometer (LCQ Deca, Thermo Electron). (a) MS analysis of pure extract by direct injection onto restricted-access column 2 in the absence of affinity protein. (b) Analysis of the same natural extract spiked with digoxin using the labelfree MS assay method as shown in Fig. 5.15.

One has to emphasize that MS also is associated with several drawbacks when it comes to bioactivity screening. First of all, the optimum, native conditions for bioactivity screening (pH 7.2, addition of 150 mM sodium chloride) are entirely incompatible with optimum conditions for MS detection which, for ESI-MS, typically require acidic pH values and the presence of organic modifiers to enhance ionization properties of the analytes. Assay development for MS-based assays therefore mainly requires the testing of different assay conditions, particularly the replacement of nonvolatile buffers with MS-compatible volatile buffers. Furthermore, it is essential to monitor ion suppression effects, for example, by the addition of system-monitoring compounds, as shown in this article. Ion suppression may very well lead to wrongful assignments of bioactivities which is particularly harmful if it leads to false negative results. Assay development should therefore also comprise the design of control experiments, e.g. omitting proteins in binding studies, addition of competitors, in order to allow an accurate assessment of the biochemical properties of bioactive substances.

Miniaturization is a key aspect when implementing MS-based assays. The high sensitivity of ESI-MS in capillary or nano-LC mode favors the use of miniaturized assay formats. Both open-tubular capillaries and chip-based designs enable the establishment of low deadvolume microfluidic assays; however, one should keep in mind that injection volumes for miniaturized analytical systems are substantially lower than in macrofluidic systems. In our experience, it is essential to integrate on-line preconcentration methods, for example, solid-phase extraction or on-column focusing of analytes, to enable larger injection volumes, to achieve the detection limits relevant for screening in a drug discovery environment.

With the development of high-resolution MS instruments such as FT-ICR MS, mass spectrometry will certainly gain in importance for studying key properties of hit and lead compounds in the early stages of drug discovery. In view of the diversity of many protein targets to be screened, it is in our opinion advisable to rely on a broad portfolio of assay formats rather than focusing on a single approach. Next to the methodologies described in this contribution, assay formats detecting protein-ligand complexes by, for example, MALDI-TOF MS should also be considered.

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