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Dernière mise à jour : Mai 2018

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SPS - Saclay Plant Sciences

The PAPPSO proteome analysis platform

The proteome is defined as the protein complement expressed by a genome. It is dynamic, in that it depends on the environment, on the developmental stage, on the type of tissue or cell considered within a given organism and even on the genotype. Proteomics includes i) the identification of the proteins expressed in biological samples, ii) the characterization of their properties like post-translational modifications, sub-cellular localization, physical interactions between them or with other molecules, abundance and iii) the analysis of their dynamics over time or in response to an environmental stimulus or to a genetic variation.

Animation showing the course of peptides and their fragments in a mass spectrometer during a proteomic analysis (https://youtu.be/K1VSYjuw6os)

Proteomics was initially based on the separation of proteins by two-dimensional electrophoresis. With the technical advances made in mass spectrometry from the 1990s onwards and the development of bottom-up proteomics, it is now possible to identify and quantify thousands of proteins without any preliminary separation step. Bottom-up proteomics is currently at the basis of much of the protein research undertaken in biology. It consists in first digesting proteins by one or more endoproteases like trypsin and then analyzing the resulting mixture of peptides by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In this technique, peptides are separated by LC and ionized by electrospray before entering the mass spectrometer where they are submitted to two stages of mass analysis separated by a stage of fragmentation. For a given peptide, four types of data are thus collected: its mass-to-charge (m/z) ratio, its signal intensity, its retention time and the m/z ratios of its fragments. The latter are used to build a fragmentation spectrum, which, together with the peptide m/z, allows identifying the peptide sequence. Quantification is based on the signal intensities collected from the peptide itself or from its fragments and isotope labelling as well as label-free methods are available.

PAPPSO (http://pappso.inra.fr) performs proteomic analyses in the framework of service provision and collaborations. It currently involves nine permanent staff members. It is equipped with four mass spectrometers adapted to different types of analyses and to different levels of sample complexity: LTQ-XL and three high-resolution instruments, LTQ-Orbitrap, Q-Exactive plus and Orbitrap Fusion Lumos. PAPPSO has been labelled by IBiSA since 2009. It is also labelled by the CNOC as Strategic INRA Platform (2009-2013) and National Strategic Platform (2013-2018).

Historically, PAPPSO arose in 2000 by merging two complementary technical platforms: one is specialized in the analysis of prokaryotes and is located in Jouy-en-Josas (UMR Micalis); the other is specialized in plant biology and is located in Gif-sur-Yvette (UMR Génétique Quantitative et Evolution - Le Moulon). These specificities do not prevent us from exploring other worlds. For example we studied the proteins of clarinet reeds, horse cartilages, not to mention pigeon droppings… That said, PAPPSO developed a unique expertise in the analysis of large experimental designs (several hundreds of samples) as well as in peptidomic and metaproteomics.  As an illustration, PAPPSO has recently carried out an experiment of genome wide association genetics on maize proteome that included over a thousand samples. Currently, the platform is also involved in a metaproteomics project aiming at analyzing the human gut microbiota of 250 individuals. To store and manage such a huge amount of data, the platform has set up a robust informatics infrastructure.

PAPPSO

The proteomics analysis of large numbers of sample reveals the differential response of genotypes to environmental variations and, combined with high density genotyping, enables the identification of loci controlling protein amounts.

In parallel with the developments made in mass spectrometry, PAPPSO also develops and disseminates in open source several bioinformatic tools adapted to big data and to the analysis of complex samples. These tools allow PAPPSO to manage the entire pipeline of data analysis, from peptide identification to protein quantification and statistical analysis. Through these developments and through the organization of regular training sessions, PAPPSO supports its users in the exploitation of their own results.

Main bioinformatic tools developed by PAPPSO:

- X!TandemPipeline (Langella et al., 2017): protein identification and inference

- MassChroQ (Valot et al., 2011): peptide quantification

- ProticDB database (Langella et al., 2013): data storage and exploitation

- R package 'MCQ': statistical analysis of proteomics data