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

Abstract

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.

Post-translational modification analysis of Saccharomyces cerevisiae histone methylation enzymes reveals phosphorylation sites of regulatory potential

“…2 and Table S3). These observations are generally consistent with absolute phosphorylation stoichiometry measurements across the human phosphoproteome, with the majority of sites existing at less than 20% occupancy (40), and studies that show that the majority of phosphosites in yeast are of ≤30% stoichiometry (41). The relatively low stoichiometry of phosphorylation in these enzymes suggests that the sites are not constitutive.…”

In‐depth and 3‐dimensional exploration of the budding yeast phosphoproteome

“…In addition, the relative prevalence of a particular phosphorylation event does not predetermine its importance. Although specialized MS applications can assess the stoichiometry of protein phosphorylation (Wu et al , 2011; Lim et al , 2017), general MS‐based phosphoproteomics does not inherently inform phosphorylation stoichiometry. Therefore, low abundant phosphoproteins that are stoichiometrically phosphorylated are often indistinguishable from very abundant proteins whose phosphorylated form represents only a small fraction of their total protein.…”

In-depth and 3-Dimensional Exploration of the Budding Yeast Phosphoproteome

“…In addition, the relative prevalence of a particular phosphorylation event does not predetermine its importance. Although specialized MS applications can assess the stoichiometry of protein phosphorylation 39 , general MS-based phosphoproteomics does not inherently inform phosphorylation stoichiometry. Therefore, lowabundant phosphoproteins that are stoichiometrically phosphorylated are often indistinguishable from very abundant proteins whose phosphorylated form represents only a small fraction of their total protein.…”