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Stable isotope labeling (SIL) has emerged as a powerful tool to measure the relative quantitative differences between samples in many differential display-type proteomic applications. However, current SIL procedures tend to suffer from the fact that one needs to decide very early in a biochemical strategy whether or not a sample will be subjected to relative quantification. Typically, the entire strategy has to be adapted to the needs of the particular quantification method chosen which might limit the range of biochemical experiments amenable to quantification. Metabolic labeling approaches, albeit very sensitive, can only be applied to studies using appropriate cell culture systems which might not necessarily be compatible with the biological system under investigation. Chemical labeling of complex protein mixtures by, e.g., isotope-coded affinity tags (ICAT), can offer great simplification of protein mixtures but is restricted by the accessibility of the often few suitable peptides (i.e. cysteine containing peptides) for both protein identification and quantification. Here, we describe a post-digest 18O-labeling method that can circumvent some of the above limitations by separating protein identification from quantification. An aliquot of all samples in a set can be used for rapid protein ID using, e.g., matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In a second step, relative quantification is performed using trypsin-catalyzed 18O incorporation into all tryptic peptides. This two-stage procedure introduces significant experimental flexibility because it enables postponement of the decision about which pairs of samples from a given set of experiments are to be compared until after the protein ID stage. In-gel digested protein quantities between 50 fmol and 15 pmol are amenable to this new method, with a dynamic range of 1:10 within one sample. Accuracy for measured relative abundances is similar to those reported for other SIL strategies (errors typically

The unorthodox two-component phosphorelay systems BvgAS and EvgAS of Bordetella pertussis and E. coli, respectively, are suitable model systems to investigate the molecular basis of signalling specificity, because, despite their high relatedness on the sequence level, they do not cross-talk to each other. We could show that the two systems belong to the obligate type of phosphorelay systems and that signalling specificity is mediated by the HPt modules of the histidine kinases and the receiver domains of the effector proteins. To gain more insight into signalling specificity on the molecular level, we started a detailed structural analysis of the respective proteins using a combination of genetic and biochemical methods including limited proteolysis and chemical modification of purified proteins and their mass spectrometrical analysis.

Most antimalarial drugs face decreased efficacy due to the emergence of resistant parasites. Therefore, the discovery of new antimalarial medicines is focused on new drugs that act by novel mechanisms and are active against different P. falciparum development stages. Screening of a focused compound library for antiparasitic activity, lead to identification of a novel class of compounds with activity against P. falciparum, 2-aminopyridines. The selected hits were validated and subjected to a lead optimization program resulting in the pre-clinical candidate MMV390048. Here we report an unbiased chemoproteomics strategy for the identification of targets of MMV390048. An analogue of MMV390048 containing a primary amine function for immobilization in a permissive position was synthesized and covalently immobilized on sepharose beads. Affinity capturing of potential target proteins from a P. falciparum blood stage extract was performed in the absence and presence of an excess of MMV390048 in the extract to delineate target proteins for which capturing is competitively inhibited. All proteins captured by the beads were quantified by isotope tagging of tryptic peptides followed by LC-MS/MS. Notably MMV390048 competitively inhibited the binding of only a single protein, P. falciparum PI4 kinase, to the beads. However, the immobilization of a drug compound via a linker may not be compatible with the binding to all of its targets. Therefore, we also performed a capturing experiment with kinobeads, which represent a combination of immobilized promiscuous ATP competitive kinase inhibitors (Bantscheff et al., 2007). As in the previous experiment, PfPI4K was the only P. falciparum protein which exhibited a reduction of bead binding upon the addition of MMV390048 to the extract. Knowledge of the target will accelerate the drug discovery process.