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    SILAC labeling (Stable Isotope Labelling by Amino acids in Cell culture):

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    A method for proteomic global quantification

    Principle

    SILAC enables the quantification of variations at the protein level between biological samples.

    Cells of interest are placed in a medium containing “light” (standard) or “heavy” amino acids (Lysine and Arginine). Heavy amino acids contain non-radioactive isotopes such as C13, N15.

    These amino acids are incorporated into proteins during cell division. Once incorporation is complete (after a few cell passages), heavy or standard medium cultivated cells are treated according to the conditions of interest (for example: infected or not, inhibited or not), then pooled together before trypsin digestion.

    During mass spectrometry analysis, heavy amino acids produce a typical mass difference between peptides. This difference is a marker of the source sample.

    The ratio between intensities of light/heavy peptides is used to obtain the relative quantification of proteins between tested conditions.

     

    Example of application

    Florence Margottin-Goguet’s team used the SILAC method in order to study modulation of proteins expression induced by HIV-1 Vpr containing virus-like-particles.

    Vpr is an auxiliary viral protein of HIV-1, which induces a cell cycle arrest in G2 via the recruitment of an ubiquitin ligase (Le Rouzic et al, Cell Cycle 2007). The team put forward the hypothesis that Vpr could induce the degradation of one or several cellular factors through this mechanism. This is how other viral auxiliary proteins inactivate host restriction factors that inhibit viral replication (Vif, Vpu or Vpx induces the degradation of APOBEC3G, tetherin/BST-2 or SAMHD1, respectively). To test this hypothesis, the team, in collaboration with the proteomics facility, chose the SILAC strategy, as it is very efficient to detect quantitative variations between samples. Wild-type Vpr or a G2 arrest-defective mutant or Vpx viral protein (third condition with incorporation of “medium” isotopes) were delivered to cells with pseudo-viral-particles.

    Proteomic analysis of the nucleus content of the cells resulted in the identification and quantification of more than 3000 proteins. Some of these proteins stabilized with wt Vpr (and not the mutant) are known to accumulate in G2, validating the procedure. Moreover, SILAC analysis led to the identification of a new cellular target degraded by Vpr: the HTLF (Helicase-like transcription factor) DNA translocase, a protein involved in the repair of DNA replication forks. This work could lead to the discovery of new mechanisms that counteract the virus (restriction mechanisms).

    The study of the mechanism of action of Vpr on HTLF has been published: Lahouassa H., Blondot ML et al. HIV-1 Vpr degrades the HLTF DNA translocase in T cells and macrophages. PNAS-2016.

     

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

    Currently, the most sensitive proteomic method proposed by our facility is the label free method described in a previous focus. However, the SILAC labeling displays specific advantages:

    • Pulse-chase SILAC allows the identification of newly synthesized proteins and the measurement of their half-life. The principle is to put cells in a heavy medium at t0, and then to take samples from this culture at different times.
    • The possibility to pool samples just before cells lysis allows decreasing technical artefacts, and it is an advantage if one whishes to study subcellular fractions (nucleus, ribosomes …) which requires extensive preparation of the samples.
    • In proteomics analysis, keratins from epithelial cells are frequent contaminants. If the study concerns this type of cells, labeling will allow distinguishing contaminants from identified proteins.

     

    In any case, a meeting upstream of your project with the facility staff is important to choose the most appropriate method.

     

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