Tardigrades have fascinated researchers for more than 300 years because of their amazing capability to undergo anhydrobiosis. In extreme states of dehydration, anhydrobiotic tardigrades undergo a metabolic dormancy, in which metabolism decreases to a non-measurable level and life comes to a reversible standstill until activity is resumed under more favourable conditions. In the anhydrobiotic (tun) state, tardigrades are extraordinary tolerant to physical extremes including high and subzero temperatures, high pressure, and extreme levels of ionizing radiation. Possessing the ability to enter this ametabolic state at any developmental stage, tardigrades are capable of surviving for a very long time and extend their lifespan significantly. Anhydrobiosis seems to be the result of dynamic processes and appears to be mediated by protective systems that prevent lethal damage. However, the survival mechanisms of tardigrades are still poorly understood. This is mainly caused by notable absence of detailed analysis concerning the proteome and genome of these organisms. FUNCRYPTA (Functional Analysis of Dynamic Processes in Cryptobiotic Tardigrades) project consisting of four research groups has been established to fill this gap by performing a broad range of investigations and analyses. As Funcrypta´s cooperation partner specialized in proteomics field we started with establishing optimal protocols for extraction of proteins from tardigrades, performing high resolution gel electrophoresis and high throughput protein identification and quantification. Since the presence of a comprehensive protein database is a prerequisite for protein identification, a M. tardigradum sequencing project has been initiated in parallel to our proteomic study by our genomic cooperation partner. The first tardigrade protein database translated from expressed sequence tags (ESTs), that have been generated by Sanger sequencing contained 3318 sequences. This protein database allowed us to develop the first proteome map of tardigrades utilizing 2D gel electrophoresis. The second protein database based on 454 sequencing with a high number of 24679 protein sequences provided us the basis for protein identification and quantification in a large scale. This resulted for the first time in a broad characterization of proteins expressed in tardigrades. More than 3000 unique proteins of M. tardigradum in three different states (early embryonic state and adults in active and anhydrobiotic states) have been identified with high sequence coverage using 1D electrophoresis in combination with high sensitive nanoLC ESI-MS/MS on a LTQ-Orbitrap mass spectrometer. Among the broad range of identified protein families, proteins known to be associated with desiccation tolerance were identified. This includes proteins with antioxidant activity, chaperones in particular heat shock proteins, aquaporins and Late Embryogenesis Abundant (LEA) proteins. Furthermore the present study provides a semi-quantitative analysis of proteins expressed in early embryonic state and adults in active and anhydrobiotic states using a label-free approach based on Exponentially Modified Protein Abundance Index (emPAI). This method allowed the classification of proteins present in one state in major and minor components and furthermore a quantitative analysis of differentially expressed proteins in each state. The semi-quantitative analysis delivered consequential results in comparing early embryonic state and adults, which will be of importance in the field of developmental biology. Using this approach we quantitatively analyzed the expressed heat shock proteins in active and tun states. The success of the analysis could be confirmed, by the published gene expression analysis of some heat shock proteins performed by our cooperation partner, which delivered similar results. The semi-quantitative analysis of active versus tun state demonstrated up-regulation of proteins in tun state that are mainly not annotated, since they are tardigrade specific and the homology search delivered no result. The functional analysis of these specific proteins in future investigations will be of major importance in regard to investigating anhydrobiosis. Analyzing the proteins that are only identified in tun state, leads to the assumption that not only proteins such as chaperones play important roles in protection mechanisms during anhydrobiosis, but also further processes and mechanisms are associated such as phosphorylation and activation of intracellular signalling cascades. Therefore optimal protocols for analyzing phosphoproteins in tardigrades have been developed and first experiments in detecting phosphoproteins on 2D gels using fluorescent dye (ProQ Diamond) have been performed. This comprehensive study from the first step of developing optimized protocol for protein extraction to the large scale protein identification and quantification builds the basis for future investigations in the field of anhydrobiotic organisms in regard to isolation and functional characterization of proteins, which are associated with protection mechanisms during anhydrobiosis. Understanding the desiccation-tolerance in anhydrobiotic tardigrades will probably enable us to develop new strategies for long-term stabilization and preservation of biological macromolecules in the future, which will be immensely important in medical field as well as in pharmaceutical industry.

Links to publications: http://www.plosone.org/article/info:doi%2F10.1371%2Fjournal.pone.0009502

http://onlinelibrary.wiley.com/doi/10.1111/j.1439-0469.2010.00608.x/suppinfo