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.