Application of SAMDI in Bioanalysis

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 kinases. 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 

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 

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. 

Since its introduction some 20 years ago, MALDI-MS has been established as a standard technique for a large variety of applications within the field of bioanalyt-ical mass spectrometry, ranging from protein identification to enzyme activity screening. Quantitative analysis has long been a challenge, but, with the use of isotopically labelled standards, it is steadily obtaining more attention. 

In contrast to established MALDI-MS techniques, DIOS-MS is a comparatively new technique. However, over the last five years, it has been gaining steadily more attention and promising results have already been obtained in all those cases, where interference from classic MALDI matrices needed to be avoided. Owing to the fact that DIOS-MS is still a juvenile technique, it is hard to predict future developments, but especially in the field of silicon modifications further promising developments can be foreseen, accompanied by new applications. 

---