Final Activity Report Summary - AMT PROTEOMICS (High throughput proteomics using accurate mass and time tags)
In the age of routine genome sequencing, biologists increasingly turn their gaze towards the molecular effectors of living organisms. Messenger ribonucleic acids (mRNA), which transcribe the genetic code, and proteins, ultimate translations of their messages, are now probed to decipher the secrets of life. While the genome can be considered more or less static, gene expressions in the form of mRNA and their translation into proteins are highly dynamic processes subject to a great variety of internal and environmental stimuli. For this reason, transcriptomics (i.e. the study of mRNA) and proteomics (i.e. the study of proteins) require broad scale assays on large numbers of samples.
Proteomics technology is rapidly evolving, being largely driven by applications in biomarkers discovery for early disease diagnostics and therapeutics. The greatest challenges in proteomics research reside in the high degree of complexity of biological samples and their extended dynamic range. According to the Human Proteome Initiative, the 21 000 human genes encode about one million different proteins. Moreover, the broad abundance range spanned by these proteins makes proteomic analyses very challenging. As an example, relative protein concentrations in human plasma cover an estimated 1010 orders of magnitudes. Measuring such diversity could be compared to using the same balance to weight an elephant and a mosquito.
Modern proteomic analyses approaches most often rely on mass spectrometry (MS) to derive protein identification and quantification. MS is a chemical analysis method designed to measure the molecular weight of chemical species. MS can also probe chemical structures in a mode known as tandem mass spectrometry or MS/MS. MS is typically used after protein digestion: A specific enzyme (e.g. trypsin) cleaves proteins at particular positions along their sequence to produces a mixture of peptides (small protein fragments). Conventional proteomics methodologies rely on protein library search after obtaining (partial) knowledge of peptide sequence through LC-MS/MS. Such methods suffer from intrinsic limitations such as analyte under sampling. In fact, detected peptides tend to be highly abundant whereas many lower-abundant ones are not consistently sampled. To overcome these issues, innovative strategies have been developed allowing peptides identification based on LC retention time and accurate mass. These methods, coined as 'Accurate mass & time (AMT) tags', have recently showed great promise for proteomics research.
Exploring new approaches to study biological samples is crucial not only in fundamental studies but also for biomedical research. With the support of a Marie Curie International Reintegration Grant funded by the European Union, the CEA Grenoble (France) has hired an expert in AMT technology, who worked previously at PNNL with R. D. Smith, the inventor of this innovative technology. With the installation of a EUR 1 million state-of-the-art instrumentation platform, and the constitution of a dedicated team, the CEA has successfully implemented the AMT technique.
Through the study of a model plant biology system, the A. thaliana chloroplast envelope, the methodology was implemented and thoroughly tested. This research provided the first chloroplast protein database; which is being put to use to determine the function of chloroplastic proteins by comparing protein profiles from diverse A. thaliana mutants (collaboration with N. Rolland and collaborators at CEA). The method has since been used to establish differential protein abundance profiles between micro dissected cholangiocarcinoma and their healthy counterparts (collaboration with C. Bréchot and collaborators at INSERM). Several biomarker candidates, which emerged from this study, are being investigated by clinicians to improve our understanding of this deadly disease.
Proteomics technology is rapidly evolving, being largely driven by applications in biomarkers discovery for early disease diagnostics and therapeutics. The greatest challenges in proteomics research reside in the high degree of complexity of biological samples and their extended dynamic range. According to the Human Proteome Initiative, the 21 000 human genes encode about one million different proteins. Moreover, the broad abundance range spanned by these proteins makes proteomic analyses very challenging. As an example, relative protein concentrations in human plasma cover an estimated 1010 orders of magnitudes. Measuring such diversity could be compared to using the same balance to weight an elephant and a mosquito.
Modern proteomic analyses approaches most often rely on mass spectrometry (MS) to derive protein identification and quantification. MS is a chemical analysis method designed to measure the molecular weight of chemical species. MS can also probe chemical structures in a mode known as tandem mass spectrometry or MS/MS. MS is typically used after protein digestion: A specific enzyme (e.g. trypsin) cleaves proteins at particular positions along their sequence to produces a mixture of peptides (small protein fragments). Conventional proteomics methodologies rely on protein library search after obtaining (partial) knowledge of peptide sequence through LC-MS/MS. Such methods suffer from intrinsic limitations such as analyte under sampling. In fact, detected peptides tend to be highly abundant whereas many lower-abundant ones are not consistently sampled. To overcome these issues, innovative strategies have been developed allowing peptides identification based on LC retention time and accurate mass. These methods, coined as 'Accurate mass & time (AMT) tags', have recently showed great promise for proteomics research.
Exploring new approaches to study biological samples is crucial not only in fundamental studies but also for biomedical research. With the support of a Marie Curie International Reintegration Grant funded by the European Union, the CEA Grenoble (France) has hired an expert in AMT technology, who worked previously at PNNL with R. D. Smith, the inventor of this innovative technology. With the installation of a EUR 1 million state-of-the-art instrumentation platform, and the constitution of a dedicated team, the CEA has successfully implemented the AMT technique.
Through the study of a model plant biology system, the A. thaliana chloroplast envelope, the methodology was implemented and thoroughly tested. This research provided the first chloroplast protein database; which is being put to use to determine the function of chloroplastic proteins by comparing protein profiles from diverse A. thaliana mutants (collaboration with N. Rolland and collaborators at CEA). The method has since been used to establish differential protein abundance profiles between micro dissected cholangiocarcinoma and their healthy counterparts (collaboration with C. Bréchot and collaborators at INSERM). Several biomarker candidates, which emerged from this study, are being investigated by clinicians to improve our understanding of this deadly disease.