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Maintenance of water homeostasis is a fundamental cellular process required by all living organisms. Here, we use the single-celled green alga Chlamydomonas reinhardtii to establish a foundational understanding of osmotic-stress signaling pathways through transcriptomics, phosphoproteomics, and functional genomics approaches. Comparison of pathways identified through these analyses with yeast and Arabidopsis allows us to infer their evolutionary conservation and divergence across these lineages. 76 genes, acting across diverse cellular compartments, were found to be important for osmotic-stress tolerance in Chlamydomonas through their functions in cytoskeletal organization, potassium transport, vesicle trafficking, mitogen-activated protein kinase and chloroplast signaling. We show that homologs for five of these genes have conserved functions in stress tolerance in Arabidopsis and reveal a novel PROFILIN-dependent stage of acclimation affecting the actin cytoskeleton that ensures tissue integrity upon osmotic stress. This study highlights the conservation of the stress response in algae and land plants, and establishes Chlamydomonas as a unicellular plant model system to dissect the osmotic stress signaling pathway.
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Arabidopsis , Chlamydomonas reinhardtii , Presión Osmótica , Transducción de Señal , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/genética , Arabidopsis/metabolismo , Arabidopsis/genética , Proteómica , Regulación de la Expresión Génica de las Plantas , Genómica , Estrés Fisiológico , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Transcriptoma , Compartimento Celular , Cloroplastos/metabolismo , MultiómicaRESUMEN
Open and practical exchange, dissemination, and reuse of specimens and data have become a fundamental requirement for life sciences research. The quality of the data obtained and thus the findings and knowledge derived is thus significantly influenced by the quality of the samples, the experimental methods, and the data analysis. Therefore, a comprehensive and precise documentation of the pre-analytical conditions, the analytical procedures, and the data processing are essential to be able to assess the validity of the research results. With the increasing importance of the exchange, reuse, and sharing of data and samples, procedures are required that enable cross-organizational documentation, traceability, and non-repudiation. At present, this information on the provenance of samples and data is mostly either sparse, incomplete, or incoherent. Since there is no uniform framework, this information is usually only provided within the organization and not interoperably. At the same time, the collection and sharing of biological and environmental specimens increasingly require definition and documentation of benefit sharing and compliance to regulatory requirements rather than consideration of pure scientific needs. In this publication, we present an ongoing standardization effort to provide trustworthy machine-actionable documentation of the data lineage and specimens. We would like to invite experts from the biotechnology and biomedical fields to further contribute to the standard.
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Hydrophobic microdomains, also known as hydrophobic patches, are essential for many important biological functions of water-soluble proteins. These include ligand or substrate binding, protein-protein interactions, proper folding after translation, and aggregation during denaturation. Unlike transmembrane domains, which are easily recognized from stretches of contiguous hydrophobic sidechains in amino acids via primary protein sequence, these three-dimensional hydrophobic patches cannot be easily predicted. The lack of experimental strategies for directly determining their locations hinders further understanding of their structure and function. Here, we posit that the small triatomic anion N3- (azide) is attracted to these patches and, in the presence of an oxidant, forms a radical that covalently modifies C-H bonds of nearby amino acids. Using two model proteins (BSA and lysozyme) and a cell-free lysate from the model higher plant Arabidopsis thaliana, we find that radical-mediated covalent azidylation occurs within buried catalytic active sites and ligand binding sites and exhibits similar behavior to established hydrophobic probes. The results herein suggest a model in which the azido radical is acting as an "affinity reagent" for nonaqueous three-dimensional protein microenvironments and is consistent with both the nonlocalized electron density of the azide moiety and the known high reactivity of azido radicals widely used in organic chemistry syntheses. We propose that the azido radical is a facile means of identifying hydrophobic microenvironments in soluble proteins and, in addition, provides a simple new method for attaching chemical handles to proteins without the need for genetic manipulation or specialized reagents.
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Azidas , Agua , Ligandos , Proteínas/química , AminoácidosRESUMEN
BACKGROUND: Accurate, high-confidence data is critical for assessing potential biothreat incidents. In a biothreat event, false-negative and -positive results have serious consequences. Worst case scenarios can result in unnecessary shutdowns or fatalities at an exorbitant monetary and psychological cost, respectively. Quantitative PCR assays for agents of interest have been successfully used for routine biosurveillance. Recently, there has been increased impetus for adoption of amplicon sequencing (AS) for biosurveillance because it enables discrimination of true positives from near-neighbor false positives, as well as broad, simultaneous detection of many targets in many pathogens in a high-throughput scheme. However, the high sensitivity of AS can lead to false positives. Appropriate controls and workflow reporting can help address these challenges. OBJECTIVES: Data reporting standards are critical to data trustworthiness. The standards presented herein aim to provide a framework for method quality assessment in biodetection. METHODS: We present a set of standards, Amplicon Sequencing Minimal Information (ASqMI), developed under the auspices of the AOAC INTERNATIONAL Stakeholder Program on Agent Detection Assays for making actionable calls in biosurveillance applications. In addition to the first minimum information guidelines for AS, we provide a controls checklist and scoring scheme to assure AS run quality and assess potential sample contamination. RESULTS: Adoption of the ASqMI guidelines will improve data quality, help track workflow performance, and ultimately provide decision makers confidence to trust the results of this new and powerful technology. CONCLUSION: AS workflows can provide robust, confident calls for biodetection; however, due diligence in reporting and controls are needed. The ASqMI guideline is the first AS minimum reporting guidance document that also provides the means for end users to evaluate their workflows to improve confidence. HIGHLIGHTS: Standardized reporting guidance for actionable calls is critical to ensuring trustworthy data.
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Proyectos de Investigación , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Duchenne Muscular Dystrophy (DMD) is an X-linked muscle disorder caused by a mutation or deletion in the dystrophin gene. In boys with DMD, muscle weakness progresses in a proximal to distal pattern, leading to gait abnormalities at all joints, in all planes of motion. Longitudinal studies are imperative to quantify changes in gait function due to DMD and are of particular importance when examining the efficacy of treatment interventions. RESEARCH QUESTION: The purpose of this study was to examine the sensitivity of the Gait Deviation Index (GDI) and Movement Analysis Profile (Gait Profile Score (GPS) and Gait Variable Score (GVS)) to quantify the longitudinal ambulatory decline in boys with DMD. A secondary aim was to quantify the effect of corticosteroid (CS) treatment. METHODS: The gait patterns of 75 boys were assessed longitudinally, 11 were steroid naïve (SN), and 64 received CS treatment. Joint kinematics were collected using either a VICON 612 or a Motion Analysis Corporation 3-D system. Representative trials were used to compute the GDI, GPS and the nine GVS for each boy for each visit. RESULTS: At baseline, GVS for the boys with DMD revealed abnormalities in all lower extremity joints and in all planes of movement compared to TD peers. GDI and GPS indices verified that the overall quality of gait in boys with DMD decreases at a significant rate with age. Boys who were steroid naïve changed at a rate 3 times greater than boys on CS in coronal plane hip motion. SIGNIFICANCE: The gait indices of GDI and GPS are able to identify changes in the quality of gait patterns in boys with DMD. Although boys on steroids had greater gait deviations than boys who were SN at baseline, the rate of decline in gait quality was slower in boys on CS.
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Distrofia Muscular de Duchenne , Masculino , Humanos , Distrofia Muscular de Duchenne/complicaciones , Fenómenos Biomecánicos , Marcha/fisiología , Articulaciones , MovimientoRESUMEN
We have developed a method for complete dissolution of whole eggs in formic acid that provides a new approach to analyzing egg biomolecules. As expected from prior work with extracted lipids, phosphatidylcholine represents the most abundant 31P NMR signal. A simplified methanol/chloroform partitioning method for separating the dissolved egg solution into metabolites, lipids and protein was performed and after ultra-high mass resolution and tandem MS fragmentation analyses several phosphatidylcholine molecules containing different fatty acid chain lengths as well as number and position of double bonds was detected. The MS based proteomic analysis further revealed 6 Gallus sequences annotated as 'uncharacterized' because they show no sequence homology with any other protein found in nature and thus, may represent proteins uniquely evolved to perform functions specific to chickens. Overall, this procedure is a rapid and facile means of characterizing in a high throughput and comprehensive manner, the molecular components of whole eggs.
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Pollos , Proteómica , Animales , Ácidos Grasos , Fosfatidilcolinas , Huevos/análisis , Cromatografía Líquida de Alta Presión/métodosRESUMEN
Background: Orthopedic treatment of flexed-knee gait consists of hamstring lengthening along with surgery at other levels. Transfer of the semitendinosus (hamstring transfer) was introduced to avoid increase of anterior pelvic tilt as well as reduce risk of recurrence. Methods: We retrospectively assessed children with spastic cerebral palsy and flexed-knee gait pre-operatively, 1 year post-operatively, and at a minimum of 7 years post-operatively. Results: The 39 patients were a mean 9.4 ± 3.4 years at the time of surgery, 20 subjects underwent hamstring transfer, and 19 subjects had hamstring lengthening with mean follow-up 9.1 years. Passive range of motion improved initially, but regressed at long term. Dynamic minimum knee flexion in stance decreased in both groups at the first post-operative study, and was maintained at final follow-up in 64-67% of patients. There was a small increase in anterior pelvic tilt at the 1-year follow-up which subsequently decreased to less than pre-operative in the hamstring lengthening group but remained mildly increased (5°) in the hamstring transfer group at final follow-up. Success in correcting stance knee flexion of the entire group was 69% of the Gross Motor Function Classification System grades I and II and 60% of the Gross Motor Function Classification System grade III subjects. Gait profile Score and sagittal knee Gait Variable Score both showed clinically important improvement after surgery and was mostly maintained long term for both groups. Lateral hamstring lengthening was beneficial in more severe patients, with minimal risk of adverse effects. Conclusion: Hamstring surgery as part of single event multi-level surgery (SEMLS) is effective in correcting flexed-knee gait in 60%-70% of patients with minimal effect on anterior pelvic tilt. There was no added advantage to hamstring transfer. Biceps Femoris lengthening may be beneficial and without significant additional risk. Level of evidence: level III.
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Over the past two decades, mass spectrometric (MS)-based proteomics technologies have facilitated the study of signaling pathways throughout biology. Nowhere is this needed more than in plants, where an evolutionary history of genome duplications has resulted in large gene families involved in posttranslational modifications and regulatory pathways. For example, at least 5% of the Arabidopsis thaliana genome (ca. 1,200 genes) encodes protein kinases and protein phosphatases that regulate nearly all aspects of plant growth and development. MS-based technologies that quantify covalent changes in the side-chain of amino acids are critically important, but they only address one piece of the puzzle. A more crucially important mechanistic question is how noncovalent interactions-which are more difficult to study-dynamically regulate the proteome's 3D structure. The advent of improvements in protein 3D technologies such as cryo-electron microscopy, nuclear magnetic resonance, and X-ray crystallography has allowed considerable progress to be made at this level, but these methods are typically limited to analyzing proteins, which can be expressed and purified in milligram quantities. Newly emerging MS-based technologies have recently been developed for studying the 3D structure of proteins. Importantly, these methods do not require protein samples to be purified and require smaller amounts of sample, opening the wider proteome for structural analysis in complex mixtures, crude lysates, and even in intact cells. These MS-based methods include covalent labeling, crosslinking, thermal proteome profiling, and limited proteolysis, all of which can be leveraged by established MS workflows, as well as newly emerging methods capable of analyzing intact macromolecules and the complexes they form. In this review, we discuss these recent innovations in MS-based "structural" proteomics to provide readers with an understanding of the opportunities they offer and the remaining challenges for understanding the molecular underpinnings of plant structure and function.
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Arabidopsis , Proteoma , Arabidopsis/genética , Microscopía por Crioelectrón , Espectrometría de Masas/métodos , Proteínas de Plantas/genética , Proteómica/métodosRESUMEN
BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disorder, that is characterized by progressive muscle degeneration and loss of ambulation between 7-13 years of age. Novel pharmacological agents targeting the genetic defects and disease mechanisms are becoming available; however, corticosteroid (CS) therapy remains the standard of care. OBJECTIVE: The purpose of this longitudinal study was to elucidate the effect of CS therapy on the rate of muscle strength and gross motor skill decline in boys with DMD and assess the sensitivity of selected outcome measures. METHODS: Eighty-four ambulatory boys with DMD (49-180 months), 70 on CS, 14 corticosteroid naïve (NCS), participated in this 8-year multi-site study. Outcomes included; isokinetic dynamometry, the Standing (STD) and Walking/Running/jumping (WRJ) dimensions of the Gross Motor Function Measure (GMFM), and Timed Function Tests (TFTs). Nonlinear mixed modeling procedures determined the rate of change with age and the influence of steroids. RESULTS: Despite CS therapy the rate of decline in strength with age was significant in all muscle groups assessed. CS therapy significantly slowed decline in knee extensor strength, as the NCS group declined at 3x the rate of the CS group. Concurrently, WRJ skills declined in the NCS group at twice the rate of the CS group. 4-stair climb and 10 meter walk/run performance was superior in the boys on CS therapy. CONCLUSION: CS therapy slowed the rate of muscle strength decline and afforded longer retention of select gross motor skills in boys on CS compared to boys who were NCS. Isokinetic dynamometry, Walk/Run/Jump skills, and select TFTs may prove informative in assessing the efficacy of new therapeutics in ambulatory boys with DMD.
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Distrofia Muscular de Duchenne , Actividades Cotidianas , Humanos , Estudios Longitudinales , Masculino , Fuerza Muscular/fisiología , Distrofia Muscular de Duchenne/tratamiento farmacológico , Caminata/fisiologíaAsunto(s)
Tobillo , Músculo Esquelético , Niño , Estudios Transversales , Humanos , Rango del Movimiento ArticularRESUMEN
BACKGROUND: In boys with DMD, muscle weakness progresses in a proximal to distal pattern, leading to compensatory gait strategies, including hyperlordosis and equinus, that increase energy cost and accelerate the loss of walking capacity. RESEARCH QUESTION: The purpose of this study was to determine the changes in the energy cost of walking that occur with disease progression and to determine the optimal normalization scheme for the longitudinal assessment of the energy cost of walking in boys with DMD. METHODS: Energy cost was assessed with the COSMED K4b2. Three normalization schemes were examined: gross energy cost (EC), net non-dimensional oxygen cost (NNcost) and speed-matched control energy cost (SMC-EC). Nonlinear mixed modeling procedures determined the rate of change with age. Linear regression was used to asses the relationship between each normalization scheme and age and body height. RESULTS: 74 boys with DMD were assessed for the energy cost of walking. Velocity decreased at a significant rate (-.00245/month, p = .03) across time; (Fig. 2), while gross EC (.003248/month, p = 0.0026), NNcost (.006155/month, p < 0.0001) and SMC-EC (.001690/month, p = 0.03) all increased significantly. Age and height were significantly associated with NNcost and SMC-EC. The sensitivity of NNcost and SMC-EC to age over time were similar, while SMC-EC was less sensitive to changes in height over time than NNcost. SIGNIFICANCE: In contrast to able-bodied peers, boys with DMD decrease their velocity while all walking energy cost measures increased over time. Both SMC-EC and NNcost proved appropriate normalization schemes for boys with DMD. Compared to gross EC, both NNcost and SMC-EC were less sensitive to changes in age over time, while SMC-EC was less sensitive to changes in height than NNcost. Therefore, both NNCost and SMC-EC are suggested normalization schemes for the longitudinal assessment of energy cost in boys with DMD.
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Distrofia Muscular de Duchenne , Estatura , Marcha , Humanos , Modelos Lineales , Masculino , CaminataRESUMEN
Coronaviruses, like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), encode a nucleotidyl transferase in the N-terminal (NiRAN) domain of the nonstructural protein (nsp) 12 protein within the RNA dependent RNA polymerase. Here we show the detection of guanosine monophosphate (GMP) and uridine monophosphate-modified amino acids in nidovirus proteins using heavy isotope-assisted mass spectrometry (MS) and MS/MS peptide sequencing. We identified lysine-143 in the equine arteritis virus (EAV) protein, nsp7, as a primary site of in vitro GMP attachment via a phosphoramide bond. In SARS-CoV-2 replicase proteins, we demonstrate nsp12-mediated nucleotidylation of nsp7 lysine-2. Our results demonstrate new strategies for detecting GMP-peptide linkages that can be adapted for higher throughput screening using mass spectrometric technologies. These data are expected to be important for a rapid and timely characterization of a new enzymatic activity in SARS-CoV-2 that may be an attractive drug target aimed at limiting viral replication in infected patients.
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Oxidative proteome damage has been implicated as a major contributor to cell death and aging. Protein damage and aging has been a particular theme of the recent research of Miroslav Radman. However, the study of how cellular proteins are damaged by oxidative processes is still in its infancy. Here we examine oxidative changes in the proteomes of four bacterial populations-wild type E. coli, two isolates from E. coli populations evolved for high levels of ionizing radiation (IR) resistance, and D. radiodurans-immediately following exposure to 3000 Gy of ionizing radiation. By a substantial margin, the most prominent intracellular oxidation events involve hydroxylation of methionine residues. Significant but much less frequent are carbonylation events on tyrosine and dioxidation events on tryptophan. A few proteins are exquisitely sensitive to targeted oxidation events, notably the active site of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in E. coli. Extensive experimental evolution of E. coli for IR resistance has decreased overall proteome sensitivity to oxidation but not to the level seen in D. radiodurans. Many observed oxidation events may reflect aspects of protein structure and/or exposure of protein surfaces to water. Proteins such as GAPDH and possibly Ef-Tu may have an evolved sensitivity to oxidation by H2O2.
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Proteoma/metabolismo , Proteoma/efectos de la radiación , Radiación Ionizante , Investigación , Escherichia coli/aislamiento & purificación , Escherichia coli/metabolismo , Escherichia coli/efectos de la radiación , Proteínas de Escherichia coli/metabolismo , Oxidación-Reducción/efectos de la radiación , Péptidos/metabolismoRESUMEN
Coronaviruses, like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), encode a nucleotidyl transferase in the N-terminal (NiRAN) domain of the nonstructural protein (nsp) 12 protein within the RNA dependent RNA polymerase. Here we show the detection of guanosine monophosphate (GMP) and uridine monophosphate-modified amino acids in nidovirus proteins using heavy isotope-assisted mass spectrometry (MS) and MS/MS peptide sequencing. We identified lysine-143 in the equine arteritis virus (EAV) protein, nsp7, as a primary site of in vitro GMP attachment via a phosphoramide bond. In SARS-CoV-2 replicase proteins, we demonstrate nsp12-mediated nucleotidylation of nsp7 lysine-2. Our results demonstrate new strategies for detecting GMP-peptide linkages that can be adapted for higher throughput screening using mass spectrometric technologies. These data are expected to be important for a rapid and timely characterization of a new enzymatic activity in SARS-CoV-2 that may be an attractive drug target aimed at limiting viral replication in infected patients.
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Comparative functional genomics offers a powerful approach to study species evolution. To date, the majority of these studies have focused on the transcriptome in mammalian and yeast phylogenies. Here, we present a novel multi-species proteomic dataset and a computational pipeline to systematically compare the protein levels across multiple plant species. Globally we find that protein levels diverge according to phylogenetic distance but is more constrained than the mRNA level. Module-level comparative analysis of groups of proteins shows that proteins that are more highly expressed tend to be more conserved. To interpret the evolutionary patterns of conservation and divergence, we develop a novel network-based integrative analysis pipeline that combines publicly available transcriptomic datasets to define co-expression modules. Our analysis pipeline can be used to relate the changes in protein levels to different species-specific phenotypic traits. We present a case study with the rhizobia-legume symbiosis process that supports the role of autophagy in this symbiotic association.
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Biología Computacional/métodos , Redes Reguladoras de Genes , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Cromatografía Liquida/métodos , Evolución Molecular , Regulación de la Expresión Génica de las Plantas , Ontología de Genes , Genómica/métodos , Filogenia , Proteínas de Plantas/genética , Plantas/clasificación , Plantas/genética , Proteoma/genética , Especificidad de la Especie , Espectrometría de Masas en Tándem/métodos , Transcriptoma/genéticaRESUMEN
[This corrects the article DOI: 10.3389/fmicb.2020.582590.].
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PURPOSE: Pharmacologic doses of corticosteroid (CS) have been shown to ameliorate the progression of Duchenne muscular dystrophy (DMD) preserving strength, pulmonary function and ambulation as well as reducing the incidence of scoliosis. However, there are serious side effects of CS, which may impact dose tolerance. The purpose of this study was to compare the magnitude of positive CS effects on patients in our clinic to those reported in the literature. METHODS: We retrospectively reviewed medical records and radiographs of 142 DMD patients who were seen between 1st January 1991 and 31st December 2017. RESULTS: In total, 101 boys met study inclusion criteria. Of these 32 were steroid naïve, 37 took the recommended dose (standard of care, SOC) of Prednisone or Deflazacort, and 32 took a lower dose (LD). Following initiation of CS, both treatment groups showed an increase in weight velocity and decrease in linear growth velocity. Although there was a trend to later loss of ambulation (LOA) in the SOC group relative to the naïve group by one year, this was not significant, however, a small subgroup of boys on Deflazacort showed a 3.4 year later LOA than the naïve group. The incidence of scoliosis was reduced from 69% in the naïve, to 41% in the LD and 47% in the SOC group. CONCLUSIONS: Although there was a reduction in the incidence of scoliosis, it was not as robust as seen elsewhere. Many published studies have inadequate data on scoliosis probably due to the lack of inclusion of orthopaedists in the study group. LEVEL OF EVIDENCE: IV.
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Ionizing radiation (IR) is lethal to most organisms at high doses, damaging every cellular macromolecule via induction of reactive oxygen species (ROS). Utilizing experimental evolution and continuing previous work, we have generated the most IR-resistant Escherichia coli populations developed to date. After 100 cycles of selection, the dose required to kill 99% the four replicate populations (IR9-100, IR10-100, IR11-100, and IR12-100) has increased from 750 Gy to approximately 3,000 Gy. Fitness trade-offs, specialization, and clonal interference are evident. Long-lived competing sub-populations are present in three of the four lineages. In IR9, one lineage accumulates the heme precursor, porphyrin, leading to generation of yellow-brown colonies. Major genomic alterations are present. IR9 and IR10 exhibit major deletions and/or duplications proximal to the chromosome replication terminus. Contributions to IR resistance have expanded beyond the alterations in DNA repair systems documented previously. Variants of proteins involved in ATP synthesis (AtpA), iron-sulfur cluster biogenesis (SufD) and cadaverine synthesis (CadA) each contribute to IR resistance in IR9-100. Major genomic and physiological changes are emerging. An isolate from IR10 exhibits protein protection from ROS similar to the extremely radiation resistant bacterium Deinococcus radiodurans, without evident changes in cellular metal homeostasis. Selection is continuing with no limit to IR resistance in evidence as our E. coli populations approach levels of IR resistance typical of D. radiodurans.
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Lipo-chitooligosaccharides (LCOs) are signaling molecules produced by rhizobial bacteria that trigger the nodulation process in legumes, and by some fungi that also establish symbiotic relationships with plants, notably the arbuscular and ecto mycorrhizal fungi. Here, we show that many other fungi also produce LCOs. We tested 59 species representing most fungal phyla, and found that 53 species produce LCOs that can be detected by functional assays and/or by mass spectroscopy. LCO treatment affects spore germination, branching of hyphae, pseudohyphal growth, and transcription in non-symbiotic fungi from the Ascomycete and Basidiomycete phyla. Our findings suggest that LCO production is common among fungi, and LCOs may function as signals regulating fungal growth and development.
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Quitina/análogos & derivados , Quitina/metabolismo , Hongos/crecimiento & desarrollo , Hongos/metabolismo , Transducción de Señal/fisiología , Ascomicetos/crecimiento & desarrollo , Basidiomycota/crecimiento & desarrollo , Quitosano , Ecología , Ácidos Grasos/metabolismo , Micorrizas/fisiología , Oligosacáridos , Rhizobium/metabolismo , Esporas Fúngicas/crecimiento & desarrollo , Simbiosis/fisiologíaRESUMEN
Recent work has begun to investigate the role of protein damage in cell death because of ionizing radiation (IR) exposure, but none have been performed on a proteome-wide basis, nor have they utilized MS (MS) to determine chemical identity of the amino acid side chain alteration. Here, we use Escherichia coli to perform the first MS analysis of IR-treated intact cells on a proteome scale. From quintuplicate IR-treated (1000 Gy) and untreated replicates, we successfully quantified 13,262 peptides mapping to 1938 unique proteins. Statistically significant, but low in magnitude (<2-fold), IR-induced changes in peptide abundance were observed in 12% of all peptides detected, although oxidative alterations were rare. Hydroxylation (+15.99 Da) was the most prevalent covalent adduct detected. In parallel with these studies on E. coli, identical experiments with the IR-resistant bacterium, Deinococcus radiodurans, revealed orders of magnitude less effect of IR on the proteome. In E. coli, the most significant target of IR by a wide margin was glyceraldehyde 3'-phosphate dehydrogenase (GAPDH), in which the thiol side chain of the catalytic Cys residue was oxidized to sulfonic acid. The same modification was detected in IR-treated human breast carcinoma cells. Sensitivity of GAPDH to reactive oxygen species (ROS) has been described previously in microbes and here, we present GAPDH as an immediate, primary target of IR-induced oxidation across all domains of life.