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    Label free proteomic analysis: stairways to the entire proteome

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    Cell reports coverA new proteomic analysis method is available at the proteomic 3P5 facility

    The 3P5 proteomic facility recently set up a new label-free proteomic analysis method at the Cochin Institute. The technique was initially developed by the JR Yates team and popularized by Matthias Mann in the Max Planck Institut für Biochemie.

    In 2016, the label free approach allowed Patrick Mayeux's team to identify more than 7500 proteins and to quantify more than 6000 of these proteins throughout erythroid differentiation, from engaged progenitors, BFU-E, to the last stage of differentiation just before enucleation, the orthochromatic stage (Gautier et al., Cell Reports, 2016). The previous proteomic analysis of erythropoiesis, published in 2009 has led to the identification of 21 differentially expressed proteins using the 2D-DIGE method (Richardson et al, Proteomics Clin Appl, 2009). The label-free method has improved the deciphering of this complex differentiation process and allowed identification of unexpected proteins that could play potential key roles in human erythropoiesis.

    In addition to the great number of quantified proteins, the label free method brings another dimension to the analyses with the absolute quantification of the proteins, either in LFQ intensity units (for Label Free Quantification), or in copy number per cell when applicable. Thus, in the study of Gautier et al., the protein quantifications were calculated in copy numbers per cell, which is a pertinent unit in such a differentiation system in which the cell volume strongly decreases, and which allows the comparison, in addition to the protein profiles, of the expression level of the proteins. For example, the authors highlight the similar level of expression of the eIF4E translation factor and of its mTORC1 pathway regulated inhibitor 4EBP1 at the beginning of the differentiation. At the last stages of differentiation, 4EBP1 expression decreases more strongly than eIF4E expression leading to a strongest expression of eIF4E compared to 4EBP1. These results suggest a strong translation control by the mTORC1 pathways at the beginning of the differentiation which strongly decreases at the end of differentiation.

    In addition to the absolute quantification, this method allows the quantification of proteins expressed at very different levels. Thus, the expression range of quantified proteins throughout erythroid differentiation expends 7 logs, from proteins expressed at only tens of copies such as MYB, to proteins expressed at several hundreds of millions of copies per cell such as for α and β globins.

    Comparing the proteomic data with already published transcriptomic data showed a modest correlation between protein and mRNA expression, as it is often the case in other models, enhancing the interest of proteomic studies.

    The label free method is now available to all researchers from the Cochin Institute and the Paris Descartes University, but also to external researchers in public and private research.

     

    Label versus label-free

    FigureIn 3 years, the 3P5 University Paris Descartes Proteomics core facility at the Cochin institute has made a significant change in strategy for global quantitative analyzes: the marking techniques (SILAC, iTRAQ and other TMT ...) used until then allowed to analyze several experimental conditions in a single injection of LC-MS measurement. Two main advantages: reduction of experimental bias and decreased analysis time. This is crucial when a single LC-MS device is available. The disadvantage of these techniques is either to have to incorporate "heavy" amino acids into cells in culture in the case of SILAC (3 conditions in multiplexing) or to chemically attach "mass labels" to the peptides at the end of sample preparation, this is the case with the iTRAQ or TMT-type reporter fragments techniques allowing multiplexing up to 10 conditions.

    However, in the space of a few years, 3P5's MS portfolio has been enriched with two high-resolution spectrometers* allowing greater availability for longer runs without labeling (Label Free Quantification or LFQ). The technical bias is then erased by the biological replicates (n = 4) and the number of conditions to be compared is no longer limited but requires the greatest stability of LC and MS performances. Moreover, the free software developed by the M. Mann & J. Cox team (ref http://dx.doi.org/10.1038/nprot.2016.136) allow comparisons of large volumes of analyzes whose possible variations can be realigned and "matched" by inter-experimental comparisons (see figure). This valuable option makes it possible to carry out identifications and quantifications by LC-MS behavioural homology whenever the spectrometer could not carry out identification by MS/MS. Thus, the number of proteins quantified per sample increases with the number of samples included in the study. The results of these comparisons are, of course, monitored by appropriate statistical tools which it is imperative to master (http://dx.doi.org/10.1038/nmeth.3901).

    The adaptation of these new tools therefore required a strong involvement of the platform's personnel, but also of the user teams to train themselves in the manipulation of large volumes of proteomic and genomic data. Indeed the number of identifications/quantifications reaches from 3000 to 8000 proteins according to the type and the cell differentiation, a depth of analysis close to the estimated exhaustivity of the genes expressed for a given cell (http://dx.doi.org/10.1038/nature13302 and http://dx.doi.org/10.1038/nature13319). Ingenuity Pathway Analysis (IPA) tools available on the platform allow valuable sorting of identification and quantification data. A "bibliographic accelerator" in a way, with an accompaniment for novices.

    * A "Qexactive" Orbitrap spectrometer co-financed by IBISA (33%), INCA (13%) and Université Paris Descartes (54%) in 2013, and a "Fusion" Orbitrap-type spectrometer co-financed with EU funds "FEDER" (50%), Cancéropole IdF (25%) and the University Paris Descartes (25%) in 2015

     

    Key point of the label free method

    • It allows an analysis of whole proteomes or sub-proteomes, without labeling. It is therefore suitable for all types of biological samples, cells or fluids
    • an unlimited number of samples can be compared
    • It allows to identify and quantify a very large number of proteins
    • Few biological material is required (only 100 µg of proteins for a global analysis of cell proteome)
    • It allows the absolute quantification in LFQ intensity for a large number of proteins simultaneously, and in some cases in copy number per cell
    • The cost of the analysis is relatively low (www.institutcochin.fr/les-plateformes/proteomique/tarifs)

     

    How do you know if this technique is suitable for your project?

    During a first meeting, the team of the 3P5 proteomics facility defines with you, the technique which is the most adapted to your problematic. They also help you to set up the preparation of your samples until dry pellet or lysate. Then the personnel of the facility take over the experiments until the statistical analysis of the results. Analysis tools are then proposed to you to extract the relevant information from your dataset.

     

    Contact

    proteomique-3p5@parisdescartes.fr

     

    Abbreviations

    3P5: Plateforme Protéomique Paris 5

    SILAC: Stable Isotope Labeling of Amino acids in Cell culture

    iTRAQ: isobaric Tag for Relative and Absolute Quantification (Applied/Sciex)

    TMT: Tandem Mass Tag (Pierce/Thermo)

    LC: Liquid Chromatography

    MS: Mass Spectrometry

     

    Figure

    Schematic of match between runs, i.e. comparison and realignments of inter-experimental runs allowed by the free software developed by the M. Mann & J. Cox team

     

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