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Purification of mRNA‐programmed translation initiation complexes suitable for mass spectrometry analysis
Author(s) -
Chicher Johana,
Simonetti Angelita,
Kuhn Lauriane,
Schaeffer Laure,
Hammann Philippe,
Eriani Gilbert,
Martin Franck
Publication year - 2015
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201400628
Subject(s) - reticulocyte , ribosome , initiation factor , mass spectrometry , chemistry , trypsin , biotinylation , translation (biology) , eukaryotic translation , messenger rna , proteomics , lysis , globin , internal ribosome entry site , tandem mass spectrometry , microbiology and biotechnology , protein biosynthesis , biochemistry , chromatography , biology , rna , enzyme , gene
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 buffer. The nanoLC‐MS/MS analysis performed on complexes assembled on β‐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.