Improved Method for Determining Absolute Phosphorylation Stoichiometry Using Bayesian Statistics and Isobaric Labeling.

Phosphorylation stoichiometry, or occupancy, is one element of phosphoproteomics that can add useful biological context (Gerber et al. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 6940-5). We previously developed a method to assess phosphorylation stoichiometry on a proteome-wide scale (Wu et al. Nat. Methods 2011, 8, 677-83). The stoichiometry calculation relies on identifying and measuring the levels of each nonphosphorylated counterpart peptide with and without phosphatase treatment. The method, however, is problematic in that low stoichiometry phosphopeptides can return negative stoichiometry values if measurement error is larger than the percent stoichiometry. Here, we have improved the stoichiometry method through the use of isobaric labeling with 10-plex TMT reagents. In this way, five phosphatase treated and five untreated samples are compared simultaneously so that each stoichiometry is represented by five ratio measurements with no missing values. We applied the method to determine basal stoichiometries of HCT116 cells growing in culture. With this method, we analyzed five biological replicates simultaneously with no need for phosphopeptide enrichment. Additionally, we developed a Bayesian model to estimate phosphorylation stoichiometry as a parameter confined to an interval between 0 and 1 implemented as an R/Stan script. Consequently, both point and interval estimates are consistent with the plausible range of values for stoichiometry. Finally, we report absolute stoichiometry measurements with credible intervals for 6772 phosphopeptides containing at least a single phosphorylation site.