Purification of mRNA-programmed translation initiation complexes suitable for mass spectrometry analysis

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Chicher, Johana | Simonetti, Angelita | Kuhn, Lauriane | Schaeffer, Laure | Hammann, Philippe | Eriani, Gilbert | Martin, Franck

Edité par CCSD ; Wiley-VCH Verlag -

International audience. Liquid Chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS) is a powerful analytical technique for the identification and mass analysis of complex protein mixtures. Here, we present a combination of methods developed for the extensive/deep proteomic analysis of purified ribosome/mRNA particles assembled in rabbit reticulocyte lysate (RRL). Ribosomes are assembled on chimeric biotinylated mRNA-DNA molecules immobilized on streptavidin-coated beads and incubated with RRL to form initiation complexes. After washing steps, the complexes are trypsin-digested directly on the beads in semi-native condition or after their elution from the beads in denaturing Laemmli buNer. The nanoLC-MS/MS analysis performed on complexes assembled on ß-globin, viral HCV, and histone H4 mRNAs revealed significant diNerences in initiation factors composition in agreement with models of translation initiation used by these diNerent types of mRNAs. Using Laemmli-denaturing condition induces release of deeply buried peptides from the ribosome and eukaryotic initiation factor 3 (eIF3) allowing the identification of the nearly complete set of ribosomal proteins.

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