Quantitative analysis of the selenoproteome for studies of the kinetics and hierarchy of selenoprotein expression in human cell lines – SELENOPROTEOME

Selenium, an element originally known for its toxicity, is now recognized to be an essential trace element and is marketed as a lifestyle drug (despite its narrow therapeutic conditions). A number of clinical and preclinical studies suggest beneficial effects of selenium related to cardiomyopathy, thyroid function, cancer and aging (which are all important societal concerns). The mechanisms of action of selenium remain in most cases elusive but the role played by selenoproteins (proteins containing selenocysteine, identified as the 21st genetically encoded amino acid used in ribosome-mediated protein synthesis) is in the centre of debate. 25 human selenoproteins have been identified on the basis of the analysis of genomic sequences using bioinformatics tools. One third of selenoproteins have never been detected in vivo and their functions are unknown. The understanding of the function and regulation of selenoproteins on the molecular level is critically dependent on the availability of adequate analytical methodology capable of studying the entire selenoproteome in-vivo simultaneously and quantitatively. To date, the only universal detection has been based on the use of gamma-radioactive 75Se labeling and gel electrophoresis autoradiography which is inappropriate for human studies and difficult in terms of handling. The canonical proteomics protocols (bottomup or shotgun) fail because of the very low selenoprotein/total protein ratio (<10-5) and the lack of specificity allowing to screen the selenoproteome fraction of the entire proteome. Hence, the simultaneous in-vivo detection, identification and quantification of all the selenoproteins which constitute the human selenoproteome requires a novel, based on a different principle approach of which the development is the principal objective of this project. The originality of the approach consists in the use of the Se-specific signal produced from selenoproteins by inductively coupled plasma sector-field mass spectrometry (ICP MS) with the absolute sensitivity of 10-15 g Se (ca. 10 attomole of selenoprotein), matrix independence and linear range of 106. The specificity to individual selenoproteins will be assured by preceding this detector by canonical proteomics separation techniques, such as 2D gel electrophoresis or multidimensional capillary chromatography using interfaces (femtosecond laser ablation or nanoflow nebulization) previously developed and patented in the coordinator's (Partner 1) laboratory in the frame of the successfully completed ACI and ANR projects. Additionally, techniques based on the use of stable (non-radioactive) tracers will be developed profiting of the isotopic resolution offered by ICP MS and the precision of the isotope ratios measurement offered by multicollector ICP MS. The instrumental developments will be carried out on selenoproteomes expressed in well defined human cell lines in culture. The design of cell culture conditions and Se depletion/supplementation will allow the validation of the approach to study the regulation of selenoproteins synthesis in mammalian cells in the Partner 2 laboratory recently recognized for its competence on selenoprotein synthesis and regulation in mammals by a young investigator grant (ATIP CNRS). The particular objective is to study the hierarchy and kinetics of expression of the individual selenoproteins. This project, based on the interdisciplinary competence in state-of-the-art analytical chemistry and selenium biochemistry aims at probing, for the first time the human selenoproteome in its entirety in vivo, which is a sine qua non condition for the discovery of biomarkers of Se-related diseases.