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

Abstract:

: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.

journal_name

Proteomics

journal_title

Proteomics

authors

Chicher J,Simonetti A,Kuhn L,Schaeffer L,Hammann P,Eriani G,Martin F

doi

10.1002/pmic.201400628

subject

Has Abstract

pub_date

2015-07-01 00:00:00

pages

2417-25

issue

14

eissn

1615-9853

issn

1615-9861

journal_volume

15

pub_type

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