Maximized quantitative phosphoproteomics allows high confidence dissection of the DNA damage signaling network

Abstract

The maintenance of genomic stability relies on DNA damage sensor kinases that detect DNA lesions and phosphorylate an extensive network of substrates. The Mec1/ATR kinase is one of the primary sensor kinases responsible for orchestrating DNA damage responses. Despite the importance of Mec1/ATR, the current network of its identified substrates remains incomplete due, in part, to limitations in mass spectrometry-based quantitative phosphoproteomics. Phosphoproteomics suffers from lack of redundancy and statistical power for generating high confidence datasets, since information about phosphopeptide identity, site-localization, and quantitation must often be gleaned from a single peptide-spectrum match (PSM). Here we carefully analyzed the isotope label swapping strategy for phosphoproteomics, using data consistency among reciprocal labeling experiments as a central filtering rule for maximizing phosphopeptide identification and quantitation. We demonstrate that the approach allows drastic reduction of false positive quantitations and identifications even from phosphopeptides with a low number of spectral matches. Application of this approach identifies new Mec1/ATR-dependent signaling events, expanding our understanding of the DNA damage signaling network. Overall, the proposed quantitative phosphoproteomic approach should be generally applicable for investigating kinase signaling networks with high confidence and depth.

Phosphoproteomics of ATR signaling in mouse testes

“…To generate a dataset enriched for early ATR-dependent signaling events that are specific to germ cells, we focused on phosphopeptides displaying consistent reduction in abundance in testes from both ATRi-treated and Rad1 cKO mice (positioned in quadrant Q2, highlighted in Figures 1F and 2A ). To exclude data points not consistently altered in both ATRi-treated and Rad1 cKO datasets, we applied a modified ‘bow-tie’ filter by excluding data points that fell outside of a fivefold interval of correlation ( Faca et al, 2020 ). Next, we applied a filter to remove phosphopeptides with inconsistent quantitative data points to increase confidence of the dataset specifically within the quadrants (see details in Materials and methods).…”

The Srs2 helicase dampens DNA damage checkpoint by recycling RPA from chromatin

“…Our model predicts that Mec1 targeting Srs2 and/or RPA could favor Srs2-mediated RPA removal from chromatin. Mec1-mediated phosphorylation of Rfa1 and Rfa2 has been shown, and potential Mec1 phosphorylation sites on Srs2 as well as the RPA chaperone Rtt105 were recently reported (44)(45)(46). Examining these phosphorylation events individually or in combination in the context of checkpoint dampening will be conducted to gain further insight into Srs2-mediated RPA regulation.…”

Phosphoproteomics reveals a distinctive Mec1/ATR signaling response upon DNA end hyper‐resection

“…SILAC phosphoproteomic data shown in Figs 1 and 2 represent the combined independent results of a “forward” (condition 1 = light/condition 2 = heavy) and a “reverse” (condition 1 = heavy/condition 2 = light) SILAC experiment. Using this experimental design, phosphorylation events that were not consistently identified in two independent, separately SILAC‐labeled yeast cultures, were filtered out, as previously described (Faca et al, 2020). All mass spectrometric data presented in this study are available through PRIDE (see data availability statement).…”