Systems Biology

Advanced mass spectrometry technology has pushed proteomic analyses to the forefront of biological and biomedical research. Limitations of proteomic approaches now often remain with sample preparations rather than with the sensitivity of protein detection. However, deciphering proteomes and their context-dependent dynamics in subgroups of tissue-embedded cells still poses a challenge, which we meet with a detailed version of our recently established protocol for cell-selective and temporally controllable metabolic labeling of proteins in Drosophila. This method is based on targeted expression of a mutated variant of methionyl-tRNA-synthetase, MetRS^L262G, which allows for charging methionine tRNAs with the non-canonical amino acid azidonorleucine (ANL) and, thus, for detectable ANL incorporation into nascent polypeptide chains.

Protein-protein interactions are at the core of a plethora of developmental, physiological and biochemical processes. Consequently, insights into the origin and evolutionary dynamics of protein-protein interactions may provide information on the constraints and dynamics of specific biomolecular circuits and their impact on the organismal phenotype.

This protocol describes how ancestral protein-protein interaction patterns can be inferred using a set of known protein interactions from phylogenetically informative species. Although this protocol focuses on protein-protein interaction data, character-state reconstructions can in general be performed with other kinds of binary data in the same way.

Leishmania is a genus of trypanosomatid protozoa and is the parasite responsible for the disease leishmaniasis. These protozoa, regulate their gene expression in an atypical way, compared to other higher eukaryotes. The regulation of gene expression is characterized by a predominance of post-transcriptional over pre-transcriptional regulatory mechanisms (Clayton, 2002). Thus proteomic analysis has proven an essential tool for understanding pathways implicated in Leishmania infectivity, host-parasite interactions, drug resistance and others. When employing a comparative proteomics analysis between different parasitic cell lines, it is essential that these lines are cultivated in exactly the same way, in the same cell density and growth phase. More importantly when cell-cycle defects are suspected, it is essential to synchronize cell-lines in the same cell-cycle phase so as to eliminate possible artifacts. This protocol describes the preparation of whole-protein samples for proteomic analysis in Leishmania donovani (L. donovani).

It has become clear that the post-embryonic growth and development of plants requires properly controlled short distance cell-to-cell communication not only through the historically well-known phytohormones, but also through secreted small peptide signals. This protocol demonstrates an example of how to isolate small peptides (< 10 daltons) from complex protein mixtures (e.g. cauliflower meristem protein extraction) for MS/MS analysis.