Final Report Summary - DISC-MS (Direct single cell mass spectrometry: A novel analytical platform for monitoring the metabolism at single-cell level)
Even synchronised and genetically identical cell populations show substantial cell-to-cell variance in a number of parameters including protein expression and physiological parameters such as stress resistance and growth rate. The goal of this European Union (EU)-supported project was to develop and apply a novel method for single cell metabolome analysis that allowed researchers to identify different phenotypes in cells that share the same genome and are present in the same microenvironment.
The motivation for this highly innovative interdisciplinary study is to gain new insight into the eukaryote cellular processes in single cells and consequently develop more accurate biological models. These in turn will provide alternative strategies for treating or preventing drug resistance and age-related disorders. Due to their lack of sensitivity, many of the currently employed metabolic analytical methods use thousands of cells but inherently average out such cell-to-cell differences by pooling cellular samples. Therefore, certain biochemical mechanisms that are naturally occurring, such as the presence of two non-genotype-derived populations that enhances the adaptability and hardiness of the population as a whole, cannot be deduced.
Therefore, I (the fellow, Dr Ibáñez) took advantage of a new cell handling system recently developed by the group of Prof. Dr Zenobi, called micro-arrays for mass spectrometry (MAMS), and incorporated it into my proposal. This technology is capable of boosting the sensitivity of traditional mass spectrometers to achieve 'single-cell' level analysis. The decision to employ the MAMS platform instead of my original concept of a microfluidic cell processing device for sample preparation and a mass spectrometric detector was made in an early stage of my project, in order to incorporate the feedback given to me by the reviewers, who evaluated my project on the application stage.
During my work, I successfully addressed one of the most difficult challenges associated with single-cell level analysis of metabolites, which is validation. By using the unicellular eukaryotic model organism Saccharomyces cerevisiae, my host (the Zenobi group at the ETH Zürich) and I designed and carried out a conceptual pilot study that demonstrates the ability of MAMS to exploit the natural occurring cell-to-cell heterogeneity to directly visualise two different phenotypes. These phenotypes are characterized by different levels of F16BP. The validation of my study concerned both the analytical methodology and the biological information, by monitoring expected cellular responses upon an environmental (the addition of a glycolytic inhibitor, e.g. 2-deoxy-d-glucose) perturbation and genetic (e.g. ?PFK2 deletion mutant) modifications.
The main outcome of this project were three peer-reviewed publications in major scientific journals and communication of the scientific outcome in major conferences (e.g. the International Mass Spectrometry Conference 2012 in Kyoto and the International workshop on metabolomics 'Technology and applications' 2011 in Bilbao, among others), as well as the establishment of my own - independent - research project at the ETH Zürich, to start my habilitation. The focus of my future research will be in the field of single-cell metabolic studies of S. cerevisiae - now enabled by the validated MAMS platform. For this purpose, this EU grant provided me with the final cornerstone prior to launching my career as an independent group leader by allowing me to establish an efficient network of collaborators such as Prof. Dr Zenobi (Switzerland, focus in analytical and technology development), Prof. Dr Petra Dittrich (Switzerlnad, focus on cell handling platform development), Prof. Dr Elisabeth Blackburn (United States (US), focus on telomere and aging research), Prof. Dr Matthias Heinemann (the Netherlands, focus on dynamic regulation processes in central metabolism), Dr Sascha Sauer (Germany, focus on glucose restriction), Dr Reinhard Dechant (Swtitzerland, focus on metabolic changes during ageing), among other past collaborators.
Project website: http://www.zenobi.ethz.ch/
The motivation for this highly innovative interdisciplinary study is to gain new insight into the eukaryote cellular processes in single cells and consequently develop more accurate biological models. These in turn will provide alternative strategies for treating or preventing drug resistance and age-related disorders. Due to their lack of sensitivity, many of the currently employed metabolic analytical methods use thousands of cells but inherently average out such cell-to-cell differences by pooling cellular samples. Therefore, certain biochemical mechanisms that are naturally occurring, such as the presence of two non-genotype-derived populations that enhances the adaptability and hardiness of the population as a whole, cannot be deduced.
Therefore, I (the fellow, Dr Ibáñez) took advantage of a new cell handling system recently developed by the group of Prof. Dr Zenobi, called micro-arrays for mass spectrometry (MAMS), and incorporated it into my proposal. This technology is capable of boosting the sensitivity of traditional mass spectrometers to achieve 'single-cell' level analysis. The decision to employ the MAMS platform instead of my original concept of a microfluidic cell processing device for sample preparation and a mass spectrometric detector was made in an early stage of my project, in order to incorporate the feedback given to me by the reviewers, who evaluated my project on the application stage.
During my work, I successfully addressed one of the most difficult challenges associated with single-cell level analysis of metabolites, which is validation. By using the unicellular eukaryotic model organism Saccharomyces cerevisiae, my host (the Zenobi group at the ETH Zürich) and I designed and carried out a conceptual pilot study that demonstrates the ability of MAMS to exploit the natural occurring cell-to-cell heterogeneity to directly visualise two different phenotypes. These phenotypes are characterized by different levels of F16BP. The validation of my study concerned both the analytical methodology and the biological information, by monitoring expected cellular responses upon an environmental (the addition of a glycolytic inhibitor, e.g. 2-deoxy-d-glucose) perturbation and genetic (e.g. ?PFK2 deletion mutant) modifications.
The main outcome of this project were three peer-reviewed publications in major scientific journals and communication of the scientific outcome in major conferences (e.g. the International Mass Spectrometry Conference 2012 in Kyoto and the International workshop on metabolomics 'Technology and applications' 2011 in Bilbao, among others), as well as the establishment of my own - independent - research project at the ETH Zürich, to start my habilitation. The focus of my future research will be in the field of single-cell metabolic studies of S. cerevisiae - now enabled by the validated MAMS platform. For this purpose, this EU grant provided me with the final cornerstone prior to launching my career as an independent group leader by allowing me to establish an efficient network of collaborators such as Prof. Dr Zenobi (Switzerland, focus in analytical and technology development), Prof. Dr Petra Dittrich (Switzerlnad, focus on cell handling platform development), Prof. Dr Elisabeth Blackburn (United States (US), focus on telomere and aging research), Prof. Dr Matthias Heinemann (the Netherlands, focus on dynamic regulation processes in central metabolism), Dr Sascha Sauer (Germany, focus on glucose restriction), Dr Reinhard Dechant (Swtitzerland, focus on metabolic changes during ageing), among other past collaborators.
Project website: http://www.zenobi.ethz.ch/