Profiling Tel1 Signaling Reveals a Non-Canonical Motif Targeting DNA Repair and Telomere Control Machineries.

The stability of the genome relies on Phosphatidyl Inositol 3-Kinase-related Kinases (PIKKs) that sense DNA damage and trigger elaborate downstream signaling responses. In S. cerevisiae, the Tel1 kinase (ortholog of human ATM) is activated at DNA double strand breaks (DSBs) and short telomeres. Despite the well-established roles of Tel1 in the control of telomere maintenance, suppression of chromosomal rearrangements, activation of cell cycle checkpoints, and repair of DSBs, the substrates through which Tel1 controls these processes remain incompletely understood. Here we performed an in depth phosphoproteomic screen for Tel1-dependent phosphorylation events. To achieve maximal coverage of the phosphoproteome, we developed a scaled-up approach that accommodates large amounts of protein extracts and chromatographic fractions. Compared to previous reports, we expanded the number of detected Tel1-dependent phosphorylation events by over 10-fold. Surprisingly, in addition to the identification of phosphorylation sites featuring the canonical motif for Tel1 phosphorylation (S/T-Q), the results revealed a novel motif (D/E-S/T) highly prevalent and enriched in the set of Tel1-dependent events. This motif is unique to Tel1 signaling and not shared with the Mec1 kinase, providing clues to how Tel1 plays specialized roles in DNA repair and telomere length control. Overall, these findings define a Tel1-signaling network targeting numerous proteins involved in DNA repair, chromatin regulation, and telomere maintenance that represents a framework for dissecting the molecular mechanisms of Tel1 action.

Multi-step control of homologous recombination via Mec1/ATR suppresses chromosomal rearrangements.

The Mec1/ATR kinase is crucial for genome stability, yet the mechanism by which it prevents gross chromosomal rearrangements (GCRs) remains unknown. Here we find that in cells with deficient Mec1 signaling, GCRs accumulate due to the deregulation of multiple steps in homologous recombination (HR). Mec1 primarily suppresses GCRs through its role in activating the canonical checkpoint kinase Rad53, which ensures the proper control of DNA end resection. Upon loss of Rad53 signaling and resection control, Mec1 becomes hyperactivated and triggers a salvage pathway in which the Sgs1 helicase is recruited to sites of DNA lesions via the 911-Dpb11 scaffolds and phosphorylated by Mec1 to favor heteroduplex rejection and limit HR-driven GCR accumulation. Fusing an ssDNA recognition domain to Sgs1 bypasses the requirement of Mec1 signaling for GCR suppression and nearly eliminates D-loop formation, thus preventing non-allelic recombination events. We propose that Mec1 regulates multiple steps of HR to prevent GCRs while ensuring balanced HR usage when needed for promoting tolerance to replication stress.

Neuromonitoring of Pediatric and Adult Extracorporeal Membrane Oxygenation Patients: The Importance of Continuous Bedside Tools in Driving Neuroprotective Clinical Care.

Extracorporeal membrane oxygenation (ECMO) is a form of temporary cardiopulmonary bypass for patients with acute respiratory or cardiac failure refractory to conventional therapy. Its usage has become increasingly widespread and while reported survival after ECMO has increased in the past 25 years, the incidence of neurological injury has not declined, leading to the pressing question of how to improve time-to-detection and diagnosis of neurological injury. The neurological status of patients on ECMO is clinically difficult to evaluate due to multiple factors including illness, sedation, and pharmacological paralysis. Thus, increasing attention has been focused on developing tools and techniques to measure and monitor the brain of ECMO patients to identify dynamic risk factors and monitor patients' neurophysiological state as a function in time. Such tools may guide neuroprotective interventions and thus prevent or mitigate brain injury. Current means to continuously monitor and prevent neurological injury in ECMO patients are rather limited; most techniques provide indirect or postinsult recognition of irreversible brain injury. This review will explore the indications, advantages, and disadvantages of standard-of-care, emerging, and investigational technologies for neurological monitoring on ECMO, focusing on bedside techniques that provide continuous assessment of neurological health.

Multi-Step Control of Homologous Recombination by Mec1/ATR Ensures Robust Suppression of Gross Chromosomal Rearrangements.

The Mec1/ATR kinase is crucial for genome stability, yet the mechanism by which it prevents gross chromosomal rearrangements (GCRs) remains unknown. Here we find that in cells with deficient Mec1 signaling, GCRs accumulate due to the deregulation of multiple steps in homologous recombination (HR). Mec1 primarily suppresses GCRs through its role in activating the canonical checkpoint kinase Rad53, which ensures the proper control of DNA end resection. Upon loss of Rad53 signaling and resection control, Mec1 becomes hyperactivated and triggers a salvage pathway in which the Sgs1 helicase is recruited to sites of DNA lesions via the 911-Dpb11 scaffolds to favor heteroduplex rejection and limit HR-driven GCR accumulation. Fusing an ssDNA recognition domain to Sgs1 bypasses the requirement of Mec1 signaling for GCR suppression and nearly eliminates D-loop formation, thus preventing non-allelic recombination events. We propose that Mec1 regulates multiple steps of HR to prevent GCRs while ensuring balanced HR usage when needed for promoting tolerance to replication stress.

Association between preoperative respiratory symptoms and perioperative respiratory adverse events in pediatric patients with positive viral testing.

STUDY OBJECTIVE: To determine the association between the presence of upper respiratory tract viral infection symptoms and occurrence of perioperative respiratory adverse events (PRAE) in children with positive viral screening, and to analyze the risk of PRAE in children with SARS-CoV-2 compared to non-SARS-CoV-2 infection. DESIGN: A prospective cohort study. SETTING: A tertiary, freestanding pediatric hospital in Dallas, Texas. PATIENTS: Children <18 years of age with positive respiratory viral testing who underwent general anesthesia. INTERVENTION: Measurement of incidence of PRAE and severe adverse events during the first 7 postoperative days. MEASUREMENTS: The primary outcome was a composite of PRAE: oxygen saturation < 90% for >5 min, supplemental oxygen for >2 h after anesthesia, laryngospasm, and bronchospasm. The secondary outcome was severe adverse events: high flow nasal cannula >6 l of oxygen per minute, admission to the ICU for escalation of respiratory support post-anesthetic, acute respiratory distress syndrome, postoperative pneumonia, cardiovascular arrest, extracorporeal life support, and death. MAIN RESULTS: In this convenience sample of 196 children, 83 were symptomatic and 113 were asymptomatic. The risk of PRAE was similar in children with active viral symptoms and asymptomatic children (risk difference: -1.9%; 95% CI: -10.9, 7.9%), but higher among children with documented fever within 48 h of the anesthetic (risk difference: 20.8%; 95% CI: 5.3, 39.7%). The multivariable adjusted odds ratio of PRAE was 0.68 (95% CI: 0.25, 1.85) for symptomatic compared to asymptomatic patients, and 0.46 (95% CI: 0.14, 1.44) for patients with SARS-CoV-2 compared to non-SARS-CoV-2 infection. CONCLUSIONS: There was no significant difference in the incidence of PRAE between symptomatic and asymptomatic children with laboratory confirmed viral respiratory infection, and between children with the Omicron variant of SARS-CoV-2 compared to non-SARS-CoV-2 respiratory viruses. However, the risk was increased in children with recent fever.

Mec1-independent activation of the Rad53 checkpoint kinase revealed by quantitative analysis of protein localization dynamics.

The replication checkpoint is essential for accurate DNA replication and repair, and maintenance of genomic integrity when a cell is challenged with genotoxic stress. Several studies have defined the complement of proteins that change subcellular location in the budding yeast Saccharomyces cerevisiae following chemically induced DNA replication stress using methyl methanesulfonate (MMS) or hydroxyurea (HU). How these protein movements are regulated remains largely unexplored. We find that the essential checkpoint kinases Mec1 and Rad53 are responsible for regulating the subcellular localization of 159 proteins during MMS-induced replication stress. Unexpectedly, Rad53 regulation of the localization of 52 proteins is independent of its known kinase activator Mec1, and in some scenarios independent of Tel1 or the mediator proteins Rad9 and Mrc1. We demonstrate that Rad53 is phosphorylated and active following MMS exposure in cells lacking Mec1 and Tel1. This noncanonical mode of Rad53 activation depends partly on the retrograde signaling transcription factor Rtg3, which also facilitates proper DNA replication dynamics. We conclude that there are biologically important modes of Rad53 protein kinase activation that respond to replication stress and operate in parallel to Mec1 and Tel1.

Hypothermia after extracorporeal cardiopulmonary resuscitation not associated with improved neurologic complications or survival in children: An analysis of the ELSO registry.

AIM: To analyze the association between hypothermia and neurologic complications among children who were treated with extracorporeal cardiopulmonary resuscitation (ECPR) using the Extracorporeal Life Support Organization (ELSO) international registry. METHODS: We conducted a retrospective, multicenter, database study utilizing ELSO data for ECPR encounters from January 1, 2011, through December 31, 2019. Exclusion criteria included multiple ECMO runs and lack of variable data. The primary exposure was hypothermia under 34 °C for greater than 24 hours. The primary outcome, determined a priori, was a composite of neurologic complications defined by ELSO registry including brain death, seizures, infarction, hemorrhage, diffuse ischemia. Secondary outcomes were mortality on ECMO and mortality prior to hospital discharge. Multivariable logistic regression determined the odds of neurologic complications, mortality on ECMO or prior to hospital discharge associated with hypothermia after adjustment for available pertinent covariables. RESULTS: Of the 2,289 ECPR encounters, no difference in odds of neurologic complications were found between the hypothermia and non-hypothermia groups (AOR 1.10, 95% CI 0.80-1.51). However, hypothermia exposure was associated with decreased odds of mortality on ECMO (AOR 0.76, 95% CI 0.59-0.97), but no difference in mortality prior to hospital discharge (AOR 0.96, 95% CI 0.76-1.21) CONCLUSION: Analysis of a large, multicenter, international dataset demonstrates that hypothermia for greater than 24 hours among children who undergo ECPR is not associated with decreased neurologic complications or mortality benefit at time of hospital discharge.

Association Between Disrupted Cerebral Autoregulation and Radiographic Neurologic Injury for Children on Extracorporeal Membrane Oxygenation: A Prospective Pilot Study.

Validation of a real-time monitoring device to evaluate the risk or occurrence of neurologic injury while on extracorporeal membrane oxygenation (ECMO) may aid clinicians in prevention and treatment. Therefore, we performed a pilot prospective cohort study of children under 18 years old on ECMO to analyze the association between cerebral blood pressure autoregulation as measured by diffuse correlation spectroscopy (DCS) and radiographic neurologic injury. DCS measurements of regional cerebral blood flow were collected on enrolled patients and correlated with mean arterial blood pressure to determine the cerebral autoregulation metric termed DCSx. The primary outcome of interest was radiographic neurologic injury on eligible computed tomography (CT) or magnetic resonance imaging (MRI) scored by a blinded pediatric neuroradiologist utilizing a previously validated scale. Higher DCSx scores, which indicate disruption of cerebral autoregulation, were associated with higher radiographic neurologic injury score (slope, 11.0; 95% confidence interval [CI], 0.29-22). Patients with clinically significant neurologic injury scores of 10 or more had higher median DCSx measures than patients with lower neurologic injury scores (0.48 vs . 0.13; p = 0.01). Our study indicates that obtaining noninvasive DCS measures for children on ECMO is feasible and disruption of cerebral autoregulation determined from DCS is associated with higher radiographic neurologic injury score.

Losing Ceci.

In this narrative medicine essay, a critical care physician's shared grief with a father who also recently experienced the death of a young child helps guide him back from his family's unimaginable loss.

Proximity Labeling Reveals Spatial Regulation of the Anaphase-Promoting Complex/Cyclosome by a Microtubule Adaptor.

The anaphase-promoting complex/cyclosome (APC/C) coordinates advancement through mitosis via temporally controlled polyubiquitination events. Despite the long-appreciated spatial organization of key events in mitosis mediated largely by cytoskeletal networks, the spatial regulation of APC/C, the major mitotic E3 ligase, is poorly understood. We describe a microtubule-resident protein, PLEKHA5, as an interactor of APC/C and spatial regulator of its activity in mitosis. Microtubule-localized proximity biotinylation tools revealed that PLEKHA5 depletion decreased APC/C association with the microtubule cytoskeleton, which prevented efficient loading of APC/C with its coactivator CDC20 and led to reduced APC/C E3 ligase activity. PLEKHA5 knockdown delayed mitotic progression, causing accumulation of APC/C substrates dependent upon the PLEKHA5-APC/C interaction in microtubules. We propose that PLEKHA5 functions as an adaptor of APC/C that promotes its subcellular localization to microtubules and facilitates its activation by CDC20, thus ensuring the timely turnover of key mitotic APC/C substrates and proper progression through mitosis.

A field guide to the proteomics of post-translational modifications in DNA repair.

All cells incur DNA damage from exogenous and endogenous sources and possess pathways to detect and repair DNA damage. Post-translational modifications (PTMs), in the past 20 years, have risen to ineluctable importance in the study of the regulation of DNA repair mechanisms. For example, DNA damage response kinases are critical in both the initial sensing of DNA damage as well as in orchestrating downstream activities of DNA repair factors. Mass spectrometry-based proteomics revolutionized the study of the role of PTMs in the DNA damage response and has canonized PTMs as central modulators of nearly all aspects of DNA damage signaling and repair. This review provides a biologist-friendly guide for the mass spectrometry analysis of PTMs in the context of DNA repair and DNA damage responses. We reflect on the current state of proteomics for exploring new mechanisms of PTM-based regulation and outline a roadmap for designing PTM mapping experiments that focus on the DNA repair and DNA damage responses.

Racial/ethnic differences in receipt of surgery among children in the United States.

BACKGROUND: It is unknown whether racial/ethnic disparities exist in surgical utilization for children. The aim, therefore, was to evaluate the odds of surgery among children in the US by race/ethnicity to test the hypothesis that minority children have less surgery. METHODS: Cross-sectional data were analyzed on children 0-18 years old from the 1999 to 2018 National Health Interview Survey, a large, nationally representative survey. The primary outcome was odds of surgery in the prior 12 months for non Latino African-American, Asian, and Latino children, compared with non Latino White children, after adjustment for relevant covariates. The National Surgical Quality Improvement Program Pediatric Dataset was used to analyze the odds of emergent/urgent surgery by race/ethnicity. RESULTS: Data for 219,098 children were analyzed, of whom 10,644 (4.9%) received surgery. After adjustment for relevant covariates, African-American (AOR, 0.54; 95% CI, 0.50-0.59), Asian (AOR, 0.39; 95% CI, 0.33-0.46), and Latino (AOR, 0.62; 95% CI, 0.57-0.67) children had lower odds of surgery than White children. Latino children were more likely to require emergent or urgent surgery (AOR, 1.71; 95% CI, 1.68-1.74). CONCLUSIONS: Latino, African-American, and Asian children have significantly lower adjusted odds of having surgery than White children in America, and Latino children were more likely to have emergent or urgent surgery. These racial/ethnic differences in surgery may reflect disparities in healthcare access which should be addressed through further research, ongoing monitoring, targeted interventions, and quality-improvement efforts. LEVEL OF EVIDENCE: II. TYPE OF STUDY: Prognosis study.

Fe-NTA Microcolumn Purification of Phosphopeptides from Immunoprecipitation (IP) Eluates for Mass Spectrometry Analysis.

Protein phosphorylation is a nearly universal signaling mechanism. To date, a number of proteomics tools have been developed to analyze phosphorylation. Phosphoproteome-wide analyses using whole cell extracts suffer from incomplete coverage, often missing phosphorylation events from low-abundance proteins. In order to increase coverage of phosphorylation sites on individual proteins of interest ("phospho-mapping"), immunoprecipitation (IP) followed by phosphoenrichment is necessary. Unfortunately, most commercially available phosphoenrichment kits are not readily scalable to the low-microgram quantities of protein present in IP eluates. Here, we describe a simple method specifically optimized for the enrichment of phosphopeptides from IP samples using an Fe-NTA based method. This method can be added downstream of any standard immunoprecipitation protocol and upstream of any MS analysis pipeline. The protocol described herein is cost effective, uses commonly available laboratory reagents, and can be used to obtain deep coverage of individual protein phosphorylation patterns, supplementary to phosphoproteomics data. Graphical abstract: Phospho-mapping workflow for a hypothetical protein of interest.

Structural basis of TRAPPIII-mediated Rab1 activation.

The GTPase Rab1 is a master regulator of the early secretory pathway and is critical for autophagy. Rab1 activation is controlled by its guanine nucleotide exchange factor, the multisubunit TRAPPIII complex. Here, we report the 3.7 Å cryo-EM structure of the Saccharomyces cerevisiae TRAPPIII complex bound to its substrate Rab1/Ypt1. The structure reveals the binding site for the Rab1/Ypt1 hypervariable domain, leading to a model for how the complex interacts with membranes during the activation reaction. We determined that stable membrane binding by the TRAPPIII complex is required for robust activation of Rab1/Ypt1 in vitro and in vivo, and is mediated by a conserved amphipathic α-helix within the regulatory Trs85 subunit. Our results show that the Trs85 subunit serves as a membrane anchor, via its amphipathic helix, for the entire TRAPPIII complex. These findings provide a structural understanding of Rab activation on organelle and vesicle membranes.

Anesthetic Complications Associated With Severe Acute Respiratory Syndrome Coronavirus 2 in Pediatric Patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is associated with high perioperative morbidity and mortality among adults. The incidence and severity of anesthetic complications in children with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unknown. We hypothesized that there would be an increased incidence of intra- and postoperative complications in children with SARS-CoV-2 infection as compared to those with negative testing. METHODS: We conducted a retrospective cohort study analyzing complications for children <18 years of age who underwent anesthesia between April 28 and September 30, 2020 at a large, academic pediatric hospital. Each child with a positive SARS-CoV-2 test within the prior 10 days was matched to a patient with a negative SARS-CoV-2 test based on American Society of Anesthesiologists (ASA) physical status, age, gender, and procedure. Children who were intubated before the procedure, underwent organ transplant surgery, or had severe COVID-19 were excluded. The primary outcome was the risk difference of a composite of intra- or postoperative respiratory complications in children positive for SARS-CoV-2 compared to those with negative testing. Secondarily, we used logistic regression to determine the odds ratio for respiratory complications before and after adjustment using propensity scores weighting to adjust for possible confounders. Other secondary outcomes included neurologic, cardiovascular, hematologic, and renal complications, unanticipated postoperative admission to the intensive care unit, length of hospital stay, and mortality. RESULTS: During the study period, 9812 general anesthetics that had a preoperative SARS-CoV-2 test were identified. Sixty encounters occurred in patients who had positive SARS-CoV-2 testing preoperatively and 51 were included for analysis. The matched controls cohort included 99 encounters. A positive SARS-CoV-2 test was associated with a higher incidence of respiratory complications (11.8% vs 1.0%; risk difference 10.8%, 95% confidence interval [CI], 1.6-19.8; P = .003). After adjustment, the odds ratio for respiratory complications was 14.37 (95% CI, 1.59-130.39; P = .02) for SARS-CoV-2-positive children as compared to controls. There was no occurrence of acute respiratory distress syndrome, postoperative pneumonia, or perioperative mortality in either group. CONCLUSIONS: Pediatric patients with nonsevere SARS-CoV-2 infection had higher rates of perianesthetic respiratory complications than matched controls with negative testing. However, severe morbidity was rare and there were no mortalities. The incidence of complications was similar to previously published rates of perianesthetic complications in the setting of an upper respiratory tract infection. This risk persisted after adjustment for preoperative upper respiratory symptoms, suggesting an increased risk in symptomatic or asymptomatic SARS-CoV-2 infection.

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

The Mec1/ATR kinase is crucial for genome maintenance in response to a range of genotoxic insults, but it remains unclear how it promotes context-dependent signaling and DNA repair. Using phosphoproteomic analyses, we uncovered a distinctive Mec1/ATR signaling response triggered by extensive nucleolytic processing (resection) of DNA ends. Budding yeast cells lacking Rad9, a checkpoint adaptor and an inhibitor of resection, exhibit a selective increase in Mec1-dependent phosphorylation of proteins associated with single-strand DNA (ssDNA) transactions, including the ssDNA-binding protein Rfa2, the translocase/ubiquitin ligase Uls1, and the Sgs1-Top3-Rmi1 (STR) complex that regulates homologous recombination (HR). Extensive Mec1-dependent phosphorylation of the STR complex, mostly on the Sgs1 helicase subunit, promotes an interaction between STR and the DNA repair scaffolding protein Dpb11. Fusion of Sgs1 to phosphopeptide-binding domains of Dpb11 strongly impairs HR-mediated repair, supporting a model whereby Mec1 signaling regulates STR upon hyper-resection to influence recombination outcomes. Overall, the identification of a distinct Mec1 signaling response triggered by hyper-resection highlights the multi-faceted action of this kinase in the coordination of checkpoint signaling and HR-mediated DNA repair.

In-depth and 3-dimensional exploration of the budding yeast phosphoproteome.

Phosphorylation is one of the most dynamic and widespread post-translational modifications regulating virtually every aspect of eukaryotic cell biology. Here, we assemble a dataset from 75 independent phosphoproteomic experiments performed in our laboratory using Saccharomyces cerevisiae. We report 30,902 phosphosites identified from cells cultured in a range of DNA damage conditions and/or arrested in distinct cell cycle stages. To generate a comprehensive resource for the budding yeast community, we aggregate our dataset with the Saccharomyces Genome Database and another recently published study, resulting in over 46,000 budding yeast phosphosites. With the goal of enhancing the identification of functional phosphorylation events, we perform computational positioning of phosphorylation sites on available 3D protein structures and systematically identify events predicted to regulate protein complex architecture. Results reveal hundreds of phosphorylation sites mapping to or near protein interaction interfaces, many of which result in steric or electrostatic "clashes" predicted to disrupt the interaction. With the advancement of Cryo-EM and the increasing number of available structures, our approach should help drive the functional and spatial exploration of the phosphoproteome.

Changes in pediatric trauma during COVID-19 stay-at-home epoch at a tertiary pediatric hospital.

BACKGROUND: Trauma is the leading cause of morbidity and mortality in the pediatric population. However, during the societal disruptions secondary to the coronavirus (COVID-19) stay-at-home regulations, there have been reported changes to the pattern and severity of pediatric trauma. We review our two-institution experience. METHODS: Pediatric trauma emergency department (ED) encounters from the National Trauma Registry for a large, tertiary, metropolitan level 1 pediatric trauma center and pediatric burn admission at the regional burn center were extracted for children less than 19 years from March 15th thru May 15th during the years 2015-2020. The primary outcome was the difference in encounters during the COVID-19 (2020) epoch versus the pre-COVID-19 epoch (2015-2019). RESULTS: There were 392 pediatric trauma encounters during the COVID-19 epoch as compared to 451, 475, 520, 460, 432 (mean 467.6) during the pre-COVID-19 epoch. Overall trauma admissions and ED trauma encounters were significantly lower (p < 0.001) during COVID-19. Burn injury admissions (p < 0.001) and penetrating trauma encounters (p = 0.002) increased during the COVID-19 epoch while blunt trauma encounters decreased (p < 0.001). Trauma occurred among more white (p = 0.01) and privately insured (p < 0.001) children, but no difference in suspected abuse, injury severity, mortality, age, or gender were detected. Sub-analysis showed significant decreases in motor vehicle crashes (p < 0.001), pedestrians struck by automobile (p < 0.001), all-terrain vehicle (ATV)/motorcross/bicycle/skateboard involved injuries (p = 0.02), falls (p < 0.001), and sports related injuries (p < 0.001). Fewer injuries occurring in the playground or home play equipment such as trampolines neared significance (p = 0.05). Interpersonal violence (assault, NAT, self-harm) was lower during the COVID-19 era (p = 0.04). For burn admissions, there was a significant increase in flame burns (p < 0.001). CONCLUSIONS: Stay-at-home regulations alter societal patterns, leading to decreased overall and blunt traumas. However, the proportion of penetrating and burn injuries increased. Owing to increased stressors and time spent at home, healthcare professionals should keep a high suspicion for abuse and neglect.

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

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.