Friday, June 26, 2009

Reply: On the evolution of protein length and phosphorylation sites

Lars just pointed out in a blog post that the average protein length of a group of proteins is a strong predictor of average number of phosphorylation sites. Although this is intuitive this is something that I honestly had not fully considered. As Lars mentions this has potential implications for some of the calculations in our recently published study on the evolution of phosphorylation in yeast species.

One potential concern relates to figure 1a. We found that, although protein phosphorylation appears to diverge quickly, there is a high conservation of the relative number of phosphosites per protein for different GO groups. Lars suggests that, at least in part, this could be due to relative differences in average protein size for these different groups that in turn is highly conserved across species.

To test this hypothesis more directly I tried to correct for differences in the average protein size of different functional groups by calculating the average number of phosphorylation sites per amino-acid, instead of psites per protein. These values were then corrected for the average number of phosphorylation sites per AA in the proteome.

As before, there is still a high cross-species correlation for the average number of psites per amino-acid for different GO groups. The correlations are only somewhat smaller than before. The individual correlation coefficients among the three species changed from: S. cerevisiae versus C. albicans – R~0.90 to 0.80; S. cerevisiae versus S. pombe – R~0.91 to 0.84; S. pombe versus C. albicans – R~0.88 to 0.83. It would seem that differences in protein length explains only a small part of the observed correlations. Results in figure 1b are also not qualitative affected by this normalization suggesting that observed differences are not due to potential changes in the average size of proteins. In fact the number of amino acids per GO group is almost perfectly correlated across species.

Another potential concern relates to the sequence based prediction of phosphorylation. As explained in the methods, one of the two approaches used to predict if a protein was phosphorylated was the sum over multiple phosphorylation site predictors for the same sequence. Given the correlation shown by Lars, could it be that, at least for one of the methods, we are mostly predicting the average protein size ? To test this I normalized the phosphorylation prediction for each S. cerevisiae protein by their length. I re-tested the predictive power of this normalized value using ROC curves and the known phosphoproteins of S. cerevisiae as postives. The AROC values changed from 0.73 to 0.68. This shows that the phosphorylation propensity is not just predicting protein size although, as expected from Lars' blog post, size alone is actually a decent predictor for phosphorylation (AROC=0.66). The normalized phosphorylation propensity does not correlate with the protein size (CC~0.05) suggesting that there might ways to improve the predictors we used.

Nature or method bias ?
Are larger proteins more likely to be phosphorylated in a cell or are they more likely to be detected in a mass-spec experiment ? It is likely that what we are observing is a combination of both effects but it would be nice to know how much of this observed correlation is due to potential MS bias. I am open to suggestions for potential tests.
This is also important for what I am planning to work on next. A while ago I had noticed that prediction of phosphorylation propensity could also predict ubiquitination and vice-versa. It is possible that they are mostly related by protein size. I will try to look at this in future posts.