Journal
PROTEOMICS
Volume 15, Issue 14, Pages 2417-2425Publisher
WILEY
DOI: 10.1002/pmic.201400628
Keywords
Cell biology; mRNA; Ribosome; RNA binding proteins; Translation initiation
Funding
- CNRS
- Universite de Strasbourg
- Investissement d'Avenir program [NetRNA ANR-10-LABX-36]
- ANR [ANR-11-SVSE802501]
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Liquid Chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS) is a powerful analyticaltechnique 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 buffer. The nanoLC-MS/MS analysis performed on complexes assembled on -globin, viral HCV, and histone H4 mRNAs revealed significant differences in initiation factors composition in agreement with models of translation initiation used by these different 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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