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Success and bottlenecks of large scale plant protein turnover measurement. Success and bottlenecks of large scale plant protein turnover measurement: a tomato fruit maturation case study
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International audience. Proteostasis is a key element among the molecular mechanisms required to maintain the equilibrium between protein synthesis and degradation during organism’s development. The protein turnover rate (PTR) is the parameter to survey to better understand proteostasis mechanisms which are associated to protein synthesis (Ks) and degradation (Kd) constants. Presently, PTR measurement based on pulse SILAC labelling are not compatible with autotroph species. Then, we developed a novel large scale proteomics strategy based on suboptimal 15N metabolic labelling to calculate Ks and Kd constants in plants. Our approach essentially requires to determine the “protein fold change” (PFC) and the “protein labelled fraction” (PLF) at several labelling kinetics time points. These two values are calculated from mass spectrometry data after adapting MassChroQ, a peptide quantification software and MCQR, an R package dedicated to the statistical analysis of proteomics quantification.First, our developments take into account the peptide 15N-modified isotopic distribution to perform robust label-free quantitation to determine the PFC for each characterised peptide/protein. Second, we used the variation of the peptide isotopic distribution to estimate the LPF and its evolution during the 15N kinetic assay. Finally, we filtered the calculated Kd constants using statistical approaches. We tested this processing pipeline in a project dedicated to study tomato fruit growth and maturation. To this end, we first setup a methodology for tomato fruit 15N labelling. Then we prepared and analysed the collected samples at different time points after the 15N starting pulse. Finally, we used the raw mass spectrometry data to test our processing pipeline. This initial application provides an excellent opportunity to improve our processing pipeline and the results, successes and bottlenecks will be detailed more extensively during this presentation.
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