A large-scale method to measure absolute protein phosphorylation stoichiometries

Abstract

The underlying functional role of protein phosphorylation is impacted by its fractional stoichiometry. Thus, a comprehensive strategy to study phosphorylation dynamics should include an assessment of site stoichiometry. Here, we developed an integrated method that relies on phosphatase treatment and stable isotope labeling to determine the absolute stoichiometries of protein phosphorylation on a large-scale. This approach requires the measurement of only a single ratio relating phosphatase- and mock-treated samples. We applied the strategy to determine stoichiometries for 5,033 phosphorylation sites in Saccharomyces cerevisiae. Stoichiometries were determined from biological triplicate experiments with good reproducibility. We validated ten sites stoichiometries representing the full range of values with an absolute quantitative approach, showing excellent agreement. Using bioinformatics, we characterized the biological properties associated with phosphorylation sites with vastly differing absolute stoichiometries.

Arginine Transcriptional Response Does Not Require Inositol Phosphate Synthesis

“…Several high throughput screens have indicated that the N-terminal end of Arg82 is phosphorylated at positions in close proximity (33)(34)(35). We tested whether these modifications have any implication for cell growth or transcriptional regulation of arginine-regulated promoters in vivo.…”

Stoichiometry of Site-specific Lysine Acetylation in an Entire Proteome

“…Mass spectrometry has been used to examine phosphorylation stoichiometry in large scale studies (14,16,17). One central issue that is addressed by these approaches is that post-translationally modified peptides and their corresponding unmodified counterparts have varying ionization efficiencies, and thus, they cannot be directly compared.…”

“…Furthermore, it is unclear how the estimates behave when the changes in PTM status across conditions are small. In the context of phosphorylation, these challenges can be addressed by treating an identical stable isotope-labeled sample with a phosphatase and measuring and comparing the increased abundance of the unphosphorylated peptide (17). The percent increase provides a measurement of the stoichiometry of a phosphosite that circumvents the need for enrichment and the requirement for a change in the level of a PTM site.…”

SSPaQ: A Subtractive Segmentation Approach for the Exhaustive Parallel Quantification of the Extent of Protein Modification at Every Possible Site

“…Finally, another elegant strategy used a combination of deuteroformaldehyde/formaldehyde stable isotope chemical labeling and alkaline phosphatase treatment [12]. The sample of interest was divided into two aliquots for treatment with phosphatase and phosphatase-free control.…”

“…Following differential chemical labeling of free amines with stable isotopes, both aliquots were recombined. Mass spectrometric analysis of the recombined mixture revealed the degree of phosphorylation by measuring the signal increase from the dephosphorylated peptide of the corresponding phosphopeptide [12]. However, this is a phosphorylation-centric method, which cannot be applied to covalent chemical modifications.…”