Sub optimal 15N metabolic labelling in plant: A new way to perform large scale protein turnovers determination using the peptides isotopic distribution

Proteostasis is defined as the processes required maintaining the equilibrium between protein synthesis and degradation. Tight regulations from gene transcription to post-translational proteins modifications of these processes are required in cell cycle and survival to balance cellular development and environmental stresses. It has been shown repeatedly that the abundance of proteins only moderately correlates with that of transcripts. While the determination of protein turnover using pulsed SILAC metabolic labelling is popular for large scale proteomics investigations. Although used with small mammals (e.g. mice), such approach remains difficult with whole organisms and almost impossible with autotroph species like plants. Alternatively, it has been proposed to use inorganic sources of 15N isotopes that could be metabolically incorporated to de novo synthetized amino acids but full 15N protein labelling is almost impossible due to the high amino acid recycling rate in plant. Instead of reaching complete 14N/15N exchange as previously done by Millar et al. 1,2 , we decide to perform a 15N metabolic pulse at very low incorporation yield (around 5 % 15N enrichment). This limited incorporation rate offers some advantages of which a limited increase of mass spectrometry signal complexity. Indeed, the 15N-labelled products moderately modify the peptide natural isotopic distribution and both labelled and unlabelled product appears as an overlapping isotopic distribution. Then, the recorded signal is extensively processed to filter out the outliers. Finally, the filtered signal is finally used to first perform label free protein quantitation which provide the "Protein Fold Change" (PFC) evolution over time. Second, the experimental peptide isotopic distribution is used to determine the apparent 15N labelling rate and the evolution of the 15N "Labelled Protein Fraction" (LPF) over the labelling time. Thanks to our local bioinformatics developments including i2MassChroQ, MassChroQ and the MCQR package, we propose an approach of MS signal processing that make it possible to determine the fold change in protein (FCP) and the labelled protein fraction (LPF). Both values (FCP & LPF) are key elements and are required to estimate the turnover rate individually for each protein 3 at the proteomic scale. Furthermore, this presentation highlights that accurate isotopic distribution measurement is possible and could be used from ordinary largescale proteomics LC-MS/MS instruments to provide an interesting alternative to determine protein turnover determination at the proteome scale