Proteomic analysis of eukaryotic translation complexes.
Tracey C. Fleischer, Connie M. Weaver, Jennifer L. Jennings, Andrew J. Link
Microbiology & Immunology, Vanderbilt School of Medicine, 1161 21st Ave South, Nashville, TN, 37232, USA
At least 10% of the characterized yeast genes play a role in protein synthesis. Since two-thirds of the yeast ORFs have not been functionally characterized, a significant subset of these novel ORFs are predicted to have roles in mRNA translation. Our goal is to carry out a rigorous, unbiased biochemical approach to identify novel translation factors using proteomics. To identify proteins associated with active mRNA/ribosome complexes, 40S, 60S, 80S, and polyribosomal complexes were purified using sucrose gradients. Potential regulatory factors associated with ribosomes were stripped from the core ribosome proteins using high salt buffers. The purified proteins from both approaches were directly and comprehensively identified using the mass spectrometry method termed Direct Analysis of Large Protein Complexes (DALPC). DALPC couples multidimensional microcapillary liquid chromatography and tandem mass spectrometry with genome-assisted data analysis. To validate the proteomic data, we chose several candidates for further characterization. Ribosome association of our putative translation factors was confirmed by immunoblotting and tandem-affinity purification followed by DALPC. Also, diploid yeast strains with corresponding homozygous null alleles were tested for defects in protein synthesis. Together, the data suggest that we have successfully applied this sensitive DALPC technology to identify putative translation factors that were not detected in gel-based studies.
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