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J Proteome Res. 2005 May-Jun;4(3):909-19.

Robust method for proteome analysis by MS/MS using an entire translated genome: demonstration on the ciliome of Tetrahymena thermophila.

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Department of Chemistry and Centre for Research in Mass Spectrometry, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3.


To improve the utility of increasingly large numbers of available unannotated and initially poorly annotated genomic sequences for proteome analysis, we demonstrate that effective protein identification can be made on a large and unannotated genome. The strategy developed is to translate the unannotated genome sequence into amino acid sequence encoding putative proteins in all six reading frames, to identify peptides by tandem mass spectrometry (MS/MS), to localize them on the genome sequence, and to preliminarily annotate the protein via a similarity search by BLAST. These tasks have been optimized and automated. Optimization to obtain multiple peptide matches in effect extends the searchable region and results in more robust protein identification. The viability of this strategy is demonstrated with the identification of 223 cilia proteins in the unicellular eukaryotic model organism Tetrahymena thermophila, whose initial genomic sequence draft was released in November 2003. To the best of our knowledge, this is the first demonstration of large-scale protein identification based on such a large, unannotated genome. Of the 223 cilia proteins, 84 have no similarity to proteins in NCBI's nonredundant (nr) database. This methodology allows identifying the locations of the genes encoding these novel proteins, which is a necessary first step to downstream functional genomic experimentation.

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