Purification of mRNA-programmed translation initiation complexes suitable for mass spectrometry analysis.
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.
Institut de Biologie Moléculaire et Cellulaire, Plateforme Protéomique Strasbourg - Esplanade, Université De Strasbourg, Strasbourg, France.
Johana
J
Chicher
Institut de Biologie Moléculaire et Cellulaire, "Architecture et Réactivité de l'ARN", Université De Strasbourg, Strasbourg, France.
Angelita
A
Simonetti
Institut de Biologie Moléculaire et Cellulaire, Plateforme Protéomique Strasbourg - Esplanade, Université De Strasbourg, Strasbourg, France.
Lauriane
L
Kuhn
Institut de Biologie Moléculaire et Cellulaire, "Architecture et Réactivité de l'ARN", Université De Strasbourg, Strasbourg, France.
Laure
L
Schaeffer
Institut de Biologie Moléculaire et Cellulaire, Plateforme Protéomique Strasbourg - Esplanade, Université De Strasbourg, Strasbourg, France.
Philippe
P
Hammann
Institut de Biologie Moléculaire et Cellulaire, "Architecture et Réactivité de l'ARN", Université De Strasbourg, Strasbourg, France.
Gilbert
G
Eriani
Institut de Biologie Moléculaire et Cellulaire, "Architecture et Réactivité de l'ARN", Université De Strasbourg, Strasbourg, France.
Franck
F
Martin
D000818:Animals; D039621:Eukaryotic Initiation Factor-3 / Q000235:genetics; D039642:Eukaryotic Initiation Factors / Q000235:genetics; D016174:Hepacivirus / Q000235:genetics; D006657:Histones / Q000235:genetics; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D010442:Peptide Chain Initiation, Translational; D040901:Proteomics / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D055544:beta-Globins / Q000235:genetics
D039621:Eukaryotic Initiation Factor-3; D039642:Eukaryotic Initiation Factors; D006657:Histones; D012333:RNA, Messenger; D012367:RNA, Viral; D055544:beta-Globins
Cell biology; RNA binding proteins; Ribosome; Translation initiation; mRNA