Project description:Our purpose is to explore the dynamics of a clinically relevant proteome by determining protein turnover, synthesis and breakdown. ]. The alteration of protein turnover represents a new type of biological information which might provide unique insight in disease pathophysiology, e.g. Alzheimer disease (AD), amyotrophic lateral sclerosis or diabetes. The use of tracer methods makes it however possible to follow protein synthesis on a proteome-scale based on the incorporation of stable isotope-labeled precursors such as 13C6-leucine. The latest mass spectrometry (MS) technologies on quantitative approach called stable isotope labeling kinetics (SILK) was applied to humans after the intravenous administration of 13C6-leucine. The SILK approach has therefore the potential to generate unique in vivo information that is not accessible using classical quantitative techniques. As a matter of fact, the concentration of a given protein in a tissue or a biological fluid is the combination of multiple events and mechanisms. The translation rate at the cellular level is obviously the initial determinant which will directly impact the presence/absence of a given protein. Translation is dependent on transcription and its genetic and epigenetic regulation. Transcriptomics is therefore one of the way to follow protein synthesis, but it is known that it reflects only partially protein concentration. In fact, many subsequent events will modify protein presence starting by post-translational maturation (which in case of pathologic mutation could lead to proteins misfolding, degradation or mis-trafficking. In our study, we are focusing on CSF proteins which originate locally from resident cells (lymphocytes...) or after compartment transfer, from central nervous system (CNS) and systemic tissues. In this work, we relied on high resolution MS (HRMS) to follow the kinetics incorporation of labelled Leucine in thousands of peptides corresponding to hundreds of proteins and their isoforms. This new approach necessitated first to develop original data processing and modelling of protein turnover which is the foundation for the SILK analysis of other types of samples, including blood and urine. The information that derived from this type of large scale analysis will help build a new vision of human physiology based on protein turnovers, and compartment transfer investigation.

Dataset Information

Protocol for high-yield production of photo-leucine-labeled proteins in Escherichia coli

Publications

Protocol for High-Yield Production of Photo-Leucine-Labeled Proteins in <i>Escherichia coli</i>.

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