Imperfect Phenotypes: Transcripts and Peptides

Source: Ghaemmaghami, S., Huh, W., Bower, K., Howson, R.W., Belle, A., Dephoure, N., O’Shea, E.K., and Welssman, J.S. Global analysis of protein expression in yeast. 2003. Nature, 425:737-41

The authors created a library of strains with TAP-tagged to an ORF. They then could use a single antibody on each strain in mid-log phase to quantify the protein abundance. Comparing these values to those for complementary transcript abundance (via microarrays) and to codon bias scores, they found that there is a significant level of correlation between the measurements. Average transcript-to-protein ratio is fairly consistent near 4,000 proteins per transcript across the range of transcripts, but the protein variability for genes with the same transcript abundance was quite high. Codon bias had a similar good ratio, with high levels of variability at the same codon score. This variation was determined not to be measurement error, as a case of 206 essential proteins were retested on the TAP-tagged library in triplicate. The TAP library was able to detect a greater number of proteins than typical LC/MS methods, due to LC/MS high abundance bias.

So once again, we find that transcript differences aren't the best proxy for understanding cellular changes in response to perturbations. They may point in the right direction but a 10-fold change in one transcript and a 5-fold in another may result in the same peptide number difference. But are proteins any better, what with post-translational modifications and activations? While it is not as common place to measure proteome changes, I'm already thinking we may need to start looking into phosphorylome (?) changes to tie it all together and get a genuine comprehensive view.

Other notes:
-80% of proteome is expressed during normal growth conditions
-tandem affinity purification (TAP) tag is colmodulin binding peptide, TEV cleavage site, two IgG binding domains
-successful integrants for 98% of all ORFs in S. cerevisiae
-tagging does not hinder, TAP can be degraded
-detected 79% of essential, 83% of products corresponding to assigned gene names
=73% of all annotated ORFs
-very abundant mRNAs generally encode for abundant proteins
=some variation due to TAP tag, as subset that was retested had higher correlation than initially found
-similar, but lower, correlation between protein abundance and codon usage as measured by codon adaptation index (CAI)
=just noise at CAI < 0.2