Longitudinal Assessment of Nasopharyngeal Biomarkers Post-COVID-19: Unveiling Persistent Markers and Severity Correlations.

SARS-CoV-19 infection provokes a variety of symptoms; most patients present mild/moderate symptoms, whereas a small proportion of patients progress to severe illness with multiorgan failure accompanied by metabolic disturbances requiring ICU-level care. Given the importance of the disease, researchers focused on identifying severity-associated biomarkers in infected patients as well as markers associated with patients suffering long-COVID. However, little is known about the presence of biomarkers that remain a few years after SARS-CoV-2 infection once the patients fully recover of the symptoms. In this study, we evaluated the presence of persistent biomarkers in the nasopharyngeal tract two years after SARS-Cov-2 infection in fully asymptomatic patients, taking into account the severity of their infection (mild/moderate and severe infections). In addition to the direct identification of several components of the Coronavirus Infection Pathway in those individuals that suffered severe infections, we describe herein 371 proteins and their associated canonical pathways that define the different adverse effects of SARS-CoV-2 infections. The persistence of these biomarkers for up to two years after infection, along with their ability to distinguish the severity of the infection endured, highlights the surprising presence of persistent nasopharyngeal exudate changes in fully recovered patients.

Development of a Prediction Model for Short-Term Remission of Patients with Crohn's Disease Treated with Anti-TNF Drugs.

Therapy with anti-tumor necrosis factor (TNF) has dramatically changed the natural history of Crohn's disease (CD). However, these drugs are not without adverse events, and up to 40% of patients could lose efficacy in the long term. We aimed to identify reliable markers of response to anti-TNF drugs in patients with CD. A consecutive cohort of 113 anti-TNF naive patients with CD was stratified according to clinical response as short-term remission (STR) or non-STR (NSTR) at 12 weeks of treatment. We compared the protein expression profiles of plasma samples in a subset of patients from both groups prior to anti-TNF therapy by SWATH proteomics. We identified 18 differentially expressed proteins (p ≤ 0.01, fold change ≥ 2.4) involved in the organization of the cytoskeleton and cell junction, hemostasis/platelet function, carbohydrate metabolism, and immune response as candidate biomarkers of STR. Among them, vinculin was one of the most deregulated proteins (p < 0.001), whose differential expression was confirmed by ELISA (p = 0.054). In the multivariate analysis, plasma vinculin levels along with basal CD Activity Index, corticosteroids induction, and bowel resection were factors predicting NSTR.

The NtrYX Two-Component System of Paracoccus denitrificans Is Required for the Maintenance of Cellular Iron Homeostasis and for a Complete Denitrification under Iron-Limited Conditions.

Denitrification consists of the sequential reduction of nitrate to nitrite, nitric oxide, nitrous oxide, and dinitrogen. Nitrous oxide escapes to the atmosphere, depending on copper availability and other environmental factors. Iron is also a key element because many proteins involved in denitrification contain iron-sulfur or heme centers. The NtrYX two-component regulatory system mediates the responses in a variety of metabolic processes, including denitrification. A quantitative proteomic analysis of a Paracoccus denitrificans NtrY mutant grown under denitrifying conditions revealed the induction of different TonB-dependent siderophore transporters and proteins related to iron homeostasis. This mutant showed lower intracellular iron content than the wild-type strain, and a reduced growth under denitrifying conditions in iron-limited media. Under iron-rich conditions, it releases higher concentrations of siderophores and displayes lower nitrous oxide reductase (NosZ) activity than the wild-type, thus leading to nitrous oxide emission. Bioinformatic and qRT-PCR analyses revealed that NtrYX is a global transcriptional regulatory system that responds to iron starvation and, in turn, controls expression of the iron-responsive regulators fur, rirA, and iscR, the denitrification regulators fnrP and narR, the nitric oxide-responsive regulator nnrS, and a wide set of genes, including the cd1-nitrite reductase NirS, nitrate/nitrite transporters and energy electron transport proteins.

Sensitivity to anti-Fas is independent of increased cathepsin D activity and adrenodoxin reductase expression occurring in NOS-3 overexpressing HepG2 cells.

Stable overexpression of endothelial nitric oxide synthase (NOS-3) in HepG2 cells (4TO-NOS) leads to increased nitro-oxidative stress and upregulation of the cell death mediators p53 and Fas. Thus, NOS-3 overexpression has been suggested as a useful antiproliferative mechanism in hepatocarcinoma cells. We aimed to identify the underlying mechanism of cell death induced by NOS-3 overexpression at basal conditions and with anti-Fas treatment. The intracellular localization of NOS-3, the nitro-oxidative stress and the mitochondrial activity were analysed. In addition, the protein expression profile in 4TO-NOS was screened for differentially expressed proteins potentially involved in the induction of apoptosis. NOS-3 localization in the mitochondrial outer membrane was not associated with changes in the respiratory cellular capacity, but was related to the mitochondrial biogenesis increase and with a higher protein expression of mitochondrial complex IV. Nitro-oxidative stress and cell death in NOS-3 overexpressing cells occurred with the expression increase of pro-apoptotic genes and a higher expression/activity of the enzymes adrenodoxin reductase mitochondrial (AR) and cathepsin D (CatD). CatD overexpression in 4TO-NOS was related to the apoptosis induction independently of its catalytic activity. In addition, CatD activity inhibition by pepstatin A was not effective in blocking apoptosis induced by anti-Fas. In summary, NOS-3 overexpression resulted in an increased sensitivity to anti-Fas induced cell death, independently of AR expression and CatD activity.

Proteomic analysis through larval development of Solea senegalensis flatfish.

The post-embryonic development of the Senegalese sole, Solea senegalensis, a flatfish of growing interest in fisheries and aquaculture, is associated with drastic morpho-physiological changes during metamorphosis. Although in the last two decades knowledge on sole culture has notably increased, especially in Southern Europe, its progress was restricted due to lack of methods to control reproduction, improve larval quality and increase juvenile disease resistance. A limited knowledge of the physiological, molecular and genetic mechanisms involved is at the base of such limitation. A proteomic study was carried out to explore the molecular events that occur during S. senegalensis ontogenesis. Protein expression changes were monitored in larvae from 5 to 21 dph by combining 2DE and protein identification with de novo MS/MS sequencing. An average of 6177 ± 282 spots was resolved in 2DE gels. Hierarchical cluster analysis of the 705 selected spots grouped them in eight patterns. Thirty-four proteins were identified and assigned biological functions including structure, metabolism highlighting energy metabolism, transport, protein folding, stress response, chromatin organization and regulation of gene expression. These changes provide a sequential description of the molecular events associated with the biochemical and biological transformations that occur during sole larval development.

Proteomic analysis in caged Mediterranean crab (Carcinus maenas) and chemical contaminant exposure in Téboulba Harbour, Tunisia.

This study uses proteomics approach to assess the toxic effects of contaminants in the Mediterranean crab (Carcinus maenas) after transplantation into Téboulba fishing harbour. High levels of aliphatic and aromatic hydrocarbons were detected in sediments. Although their effects on vertebrates are well described, little is known about their early biological effects in marine invertebrates under realistic conditions. Protein expression profiles of crabs caged for 15, 30 and 60 days were compared to unexposed animals. Nineteen proteins with significant expression differences were identified by capLC-µESI-IT MS/MS and homology search on databases. Differentially expressed proteins were assigned to five different categories of biological function including: (1) chitin catabolism, (2) proteolysis, (3) exoskeleton biosynthesis, (4) protein folding and stress response, and (5) transport. The proteins showing major expression changes in C. maenas after different caging times may be considered as novel molecular biomarkers for effectively biomonitoring aquatic environment contamination.

Dysregulation of Lipid Metabolism Serves as A Link Between Alzheimer's and Cardiovascular Disease, As Witnessed in A Cross-Sectional Study.

Cardiovascular risk factors and established cardiovascular disease (CVD) increase the risk of suffering dementia of the Alzheimer's type (DAT). Here, we set out to define specific molecular profiles of CVD in patients with DAT to better understand its relationship, to unravel the mechanisms underlying the high risk of developing DAT in CVD patients and to define new markers of early disease. Plasma samples from patients with DAT, with and without CVD, were analyzed through a multiomics approach, with integration of metabolomics and proteomics datasets using the OmicsNet web-based tool. Metabolomics results showed an enrichment in lipids and lipid-like molecules. Similarly, the most significant cluster identified through proteomics was formed by 5 proteins related to lipoprotein and cholesterol metabolism. After integration and functional enrichment, glycerolipid metabolism, fatty acid degradation and sphingolipid metabolism were among the most significant functions. Finally, differential expression of ABCA1 and APOH proteins was verified, in an independent cohort also including controls and patients with CVD alone. Both proteins positively correlated with phospho-Tau (181), a classical hallmark of DAT. Different molecular profiles exist in patients with DAT, with and without CVD, with exacerbated alterations in patients in which DAT and CVD co-exist. This information may help to define biomarkers like ABCA1 and APOH that identify patients with cardiovascular dysfunction that are at high risk of developing DAT. Such markers will allow more personalized interventions to be selected, a further step towards precision medicine for individuals whose molecular profiles indicate a distinct response to the same management strategies.

Proteomic analysis of granulomas from cattle and pigs naturally infected with Mycobacterium tuberculosis complex by MALDI imaging.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has recently gained prominence for its ability to provide molecular and spatial information in tissue sections. This technology has the potential to uncover novel insights into proteins and other molecules in biological and immunological pathways activated along diseases with a complex host-pathogen interaction, such as animal tuberculosis. Thus, the present study conducted a data analysis of protein signature in granulomas of cattle and pigs naturally infected with the Mycobacterium tuberculosis complex (MTC), identifying biological and immunological signaling pathways activated throughout the disease. Lymph nodes from four pigs and four cattle, positive for the MTC by bacteriological culture and/or real-time PCR, were processed for histopathological examination and MALDI-MSI. Protein identities were assigned using the MaTisse database, and protein-protein interaction networks were visualized using the STRING database. Gene Ontology (GO) analysis was carried out to determine biological and immunological signaling pathways in which these proteins could participate together with Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Distinct proteomic profiles between cattle and pig granulomas were displayed. Noteworthy, the GO analysis revealed also common pathways among both species, such as "Complement activation, alternative pathway" and "Tricarboxylic acid cycle", which highlight pathways that are conserved among different species infected by the MTC. In addition, species-specific terms were identified in the current study, such as "Natural killer cell degranulation" in cattle or those related to platelet and neutrophil recruitment and activation in pigs. Overall, this study provides insights into the immunopathogenesis of tuberculosis in cattle and pigs, opening new areas of research and highlighting the importance, among others, of the complement activation pathway and the regulation of natural killer cell- and neutrophil-mediated immunity in this disease.

Superior metabolic improvement of polycystic ovary syndrome traits after GLP1-based multi-agonist therapy.

Polycystic ovary syndrome (PCOS) is a heterogeneous condition, defined by oligo-/anovulation, hyper-androgenism and/or polycystic ovaries. Metabolic complications are common in patients suffering PCOS, including obesity, insulin resistance and type-2 diabetes, which severely compromise the clinical course of affected women. Yet, therapeutic options remain mostly symptomatic and of limited efficacy for the metabolic and reproductive alterations of PCOS. We report here the hormonal, metabolic and gonadal responses to the glucagon-like peptide-1 (GLP1)-based multi-agonists, GLP1/Estrogen (GLP1/E), GLP1/gastric inhibitory peptide (GLP1/GIP) and GLP1/GIP/Glucagon, in two mouse PCOS models, with variable penetrance of metabolic and reproductive traits, and their comparison with metformin. Our data illustrate the superior efficacy of GLP1/E vs. other multi-agonists and metformin in the management of metabolic complications of PCOS; GLP1/E ameliorates also ovarian cyclicity in an ovulatory model of PCOS, without direct estrogenic uterotrophic effects. In keeping with GLP1-mediated brain targeting, quantitative proteomics reveals changes in common and distinct hypothalamic pathways in response to GLP1/E between the two PCOS models, as basis for differential efficiency. Altogether, our data set the basis for the use of GLP1-based multi-agonists, and particularly GLP1/E, in the personalized management of PCOS.

Biochemical responses in seabream (Sparus aurata) caged in-field or exposed to benzo(a)pyrene and paraquat. Characterization of glutathione S-transferases.

Gilthead seabream (Sparus aurata) specimens were caged in-field at the Téboulba harbour or exposed to benzo(a)pyrene [B(a)P] or to paraquat [PQ] plus B(a)P, and several biochemical biomarker responses were investigated. Antioxidant enzymes, such as glutathione peroxidase, catalase and glutathione reductase, significantly increased in the in-field and B(a)P+PQ exposures, but were only moderately affected by B(a)P alone. Glucose-6-phosphate and 6-phosphogluconate dehydrogenases significantly diminished after in-field exposure. Different responses with biotransformation enzymes were observed: the P4501A-associated EROD activity was highly induced in response to B(a)P and B(a)P+PQ exposures, while total activity of the glutathione S-transferase (GST) was similar to control. However, after purification of the GST proteins by affinity chromatograpy and analysis by two-dimensional electrophoresis, nineteen highly reproducible isoforms were resolved. In addition, some of reproducible isoforms showed different and specific expression patterns in response to contaminants. Thus, proteomic analysis of the purified GST subunits is a reliable tool for ecotoxicological research, useful in polluted marine ecosystem as an effective biomarker of contamination.

Data Processing and Analysis in Mass Spectrometry-Based Metabolomics.

Metabolomics is the latest of the omics sciences. It attempts to measure and characterize metabolites-small chemical compounds <1500 Da-on cells, tissue, or biofluids, which are usually products of biological reactions. As metabolic reactions are closer to the phenotype, metabolomics has emerged as an attractive science for various areas of research, including personalized medicine. However, due to the complexity of data obtained and the absence of curated databases for metabolite identification, data processing is the major bottleneck in this area since most technicians lack the required bioinformatics expertise to process datasets in a reliable and fast manner. The aim of this chapter is to describe the available tools for data processing that makes an inexperienced researcher capable of obtaining reliable results without having to undergo through huge parametrization steps.

The thrombus proteome in stroke reveals a key role of the innate immune system and new insights associated with its etiology, severity, and prognosis.

BACKGROUND: Nowadays little is known about the molecular profile of the occluding thrombus of patients with ischemic stroke. OBJECTIVES: To analyze the proteomic profile of thrombi in patients who experienced an ischemic stroke in order to gain insights into disease pathogenesis. METHODS: Thrombi from an exploratory cohort of patients who experienced a stroke were obtained by thrombectomy and analyzed by sequential window acquisition of all theoretical spectra-mass spectrometry. Unsupervised k-means clustering analysis was performed to stratify patients who experienced a stroke. The proteomic profile was associated with both the neurological function (National Institute of Health Stroke Scale [NIHSS]) and the cerebral involvement (Alberta Stroke Program Early CT Score [ASPECTS]) prior to thrombectomy and the clinical status of patients at 3 months using the modified Rankin Scale. In an independent cohort of 210 patients who experienced a stroke, the potential role of neutrophils in stroke severity was interrogated. RESULTS: Proteomic analysis identified 580 proteins in thrombi, which were stratified into 4 groups: hemostasis, proteasome and neurological diseases, structural proteins, and innate immune system and neutrophils. The thrombus proteome identified 3 clusters of patients with distinctive severity, prognosis, and etiology of the stroke. A protein signature clearly distinguished atherothrombotic and cardioembolic strokes. Several proteins were significantly correlated with the severity of the stroke (NIHSS and ASPECTS). Functional proteomic analysis highlighted the prominent role of neutrophils in stroke severity. This was in line with the association of neutrophil activation markers and count with NIHSS, ASPECTS, and the modified Rankin Scale score 90 days after the event. CONCLUSION: The use of sequential window acquisition of all theoretical spectra-mass spectrometry in thrombi from patients who experienced an ischemic stroke has provided new insights into pathways and players involved in its etiology, severity, and prognosis. The prominent role of the innate immune system identified might pave the way for the development of new biomarkers and therapeutic approaches in this disease.

Hepatic proteome changes in Solea senegalensis exposed to contaminated estuarine sediments: a laboratory and in situ survey.

Assessing toxicity of contaminated estuarine sediments poses a challenge to ecotoxicologists due to the complex geochemical nature of sediments and to the combination of multiple classes of toxicants. Juvenile Senegalese soles were exposed for 14 days in the laboratory and in situ (field) to sediments from three sites (a reference plus two contaminated) of a Portuguese estuary. Sediment characterization confirmed the combination of metals, polycyclic aromatic hydrocarbons and organochlorines in the two contaminated sediments. Changes in liver cytosolic protein regulation patterns were determined by a combination of two-dimensional electrophoresis with de novo sequencing by tandem mass spectrometry. From the forty-one cytosolic proteins found to be deregulated, nineteen were able to be identified, taking part in multiple cellular processes such as anti-oxidative defence, energy production, proteolysis and contaminant catabolism (especially oxidoreductase enzymes). Besides a clear distinction between animals exposed to the reference and contaminated sediments, differences were also observed between laboratory- and in situ-tested fish. Soles exposed in the laboratory to the contaminated sediments failed to induce, or even markedly down-regulated, many proteins, with the exception of a peroxiredoxin (an anti-oxidant enzyme) and a few others, when compared to reference fish. In situ exposure to the contaminated sediments revealed significant up-regulation of basal metabolism-related enzymes, comparatively to the reference condition. Down-regulation of basal metabolism enzymes, related to energy production and gene transcription, in fish exposed in the laboratory to the contaminated sediments, may be linked to sediment-bound contaminants and likely compromised the organisms' ability to deploy adequate responses against insult.

Discriminator for Cutaneous Leishmaniasis Using MALDI-MSI in a Murine Model.

Cutaneous leishmaniasis is a skin disease caused by flagellate protozoa of the genus Leishmania and transmitted by sandflies of the genus Lutzomyia. Around 1 million new cases occur in the world annually, with a total of 12 million people affected, mainly in rural areas with low access to health services and adequate treatments. In the area of the Americas, Colombia has one of the highest infection rates after Brazil. Topical treatments with pentamidine isethionate (PMD) present an attractive alternative due to their ease of application and low costs. However, cutaneous leishmaniasis lesions present nodules with seropurulent exudate that, when drying, form hyperkeratotic lesions, hindering the effective penetration of drugs for their treatment. The use of molecular histology techniques, such as MALDI-MSI, allow in situ evaluation of the penetration of the treatment to the sections of the dermis where the disease-causing parasite resides. However, the large volume of information generated makes it impossible to process it manually. Machine learning techniques allow the unsupervised processing of large amounts of information, generating prediction models for the classification of new information. This work proposes a low-cost method to generate cutaneous leishmaniasis detection and classification models using MALDI-MSI images taken from murine models. The proposed models allow a 95% efficiency when separating healthy samples from infected samples and an effectiveness of 67% when separating effectively treated samples from unsuccessfully treated samples.

Omic approaches in environmental issues.

Biomonitoring requires the application of batteries of different biomarkers, as environmental contaminants induce multiple responses in organisms that are not necessarily correlated. Omic technologies were proposed as an alternative to conventional biomarkers since these techniques quantitatively monitor many biological molecules in a high-throughput manner and thus provide a general appraisal of biological responses altered by exposure to contaminants. As the studies using omic technologies increase, it is becoming clear that any single omic approach may not be sufficient to characterize the complexity of ecosystems. This work aims to provide a preliminary working scheme for the use of combined transcriptomic and proteomic methodologies in environmental biomonitoring. There are difficulties in working with nonmodel organisms as bioindicators when combining several omic approaches. As a whole, our results with heterologous microarrays in M. spretus and suppressive subtractive hybridization (SSH) in P. clarkii indicated that animals sustaining a heavy pollution burden exhibited an enhanced immune response and/or cell apoptosis. The proteomic studies, although preliminary, provide a holistic insight regarding the manner by which pollution shifts protein intensity in two-dimensional gel electrophoresis (2-DE), completing the transcriptomic approach. In our study, the sediment element concentration was in agreement with the intensity of protein expression changes in C. maenas crabs. In conclusion, omics are useful technologies in addressing environmental issues and the determination of contamination threats.

Redox and global interconnected proteome changes in mice exposed to complex environmental hazards surrounding Doñana National Park.

Natural environments are receiving an increasing number of contaminants. Therefore, the evaluation and identification of early responses to pollution in these complex habitats is an urgent and challenging task. Doñana National Park (DNP, SW Spain) has been widely used as a model area for environmental studies because, despite its strictly protected core, it is surrounded by numerous threat sources from agricultural, mining and industrial activities. Since many pollutants often induce oxidative stress, redox proteomics was used to detect redox-based variations within the proteome of Mus spretus mice captured in DNP and the surrounding areas. Functional analysis showed that most differentially oxidized proteins are involved in the maintenance of homeostasis, by eliciting mechanisms to respond to toxic substances and oxidative stress, such as antioxidant and biotransformation processes, immune and inflammatory responses, and blood coagulation. Furthermore, changes in the overall protein abundance were also analysed by label-free quantitative proteomics. The upregulation of phase I and II biotransformation enzymes in mice from Lucio del Palacio may be an alert for organic pollution in the area located at the heart of DNP. Metabolic processes involved in protein turnover (proteolysis, amino acid catabolism, new protein biosynthesis and folding) were activated in response to oxidative damage to these biomolecules. Consequently, aerobic respiratory metabolism increased to address the greater ATP demands. Alterations of cholesterol metabolism that could cause hepatic steatosis were also detected. The proteomic detection of globally altered metabolic and physiological processes offers a complete view of the main biological changes caused by environmental pollution in complex habitats.

A multicentric study to evaluate the use of relative retention times in targeted proteomics.

Despite the maturity reached by targeted proteomic strategies, reliable and standardized protocols are urgently needed to enhance reproducibility among different laboratories and analytical platforms, facilitating a more widespread use in biomedical research. To achieve this goal, the use of dimensionless relative retention times (iRT), defined on the basis of peptide standard retention times (RT), has lately emerged as a powerful tool. The robustness, reproducibility and utility of this strategy were examined for the first time in a multicentric setting, involving 28 laboratories that included 24 of the Spanish network of proteomics laboratories (ProteoRed-ISCIII). According to the results obtained in this study, dimensionless retention time values (iRTs) demonstrated to be a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups both intra- and inter-laboratories. iRT values also showed very low variability over long time periods. Furthermore, parallel quantitative analyses showed a high reproducibility despite the variety of experimental strategies used, either MRM (multiple reaction monitoring) or pseudoMRM, and the diversity of analytical platforms employed. BIOLOGICAL SIGNIFICANCE: From the very beginning of proteomics as an analytical science there has been a growing interest in developing standardized methods and experimental procedures in order to ensure the highest quality and reproducibility of the results. In this regard, the recent (2012) introduction of the dimensionless retention time concept has been a significant advance. In our multicentric (28 laboratories) study we explore the usefulness of this concept in the context of a targeted proteomics experiment, demonstrating that dimensionless retention time values is a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups.

Using environmental proteomics to assess pollutant response of Carcinus maenas along the Tunisian coast.

Biochemical responses to pollutants were studied at four Tunisia littoral sites using Carcinus maenas as a bioindicator. Proteomic analysis was used to assess the global impact of complex pollution mixtures, and to provide new biomarkers and basic insights into pollutant toxicity. Metal contents and metallothionein levels followed a gradient based on sampling sites: Bizerte ≫ Teboulba > Gargour~Mahres. Approximately 900 and 700 spots were resolved in digestive glands and gills, respectively. Gills from Bizerte animals had the maximum number of altered spots, mostly upregulated. In other locations, the number of altered spots in gills decreased in parallel to total metals in in the following order: Teboulba > Gargour > Mahres (mostly downregulated). Out of the 39 spots excised, ten proteins were identified in digestive glands and eight in gills. Digestive glands of Bizerte crabs had higher levels of ferritin, three vitellogenin forms and mannose-binding protein, while Gargour crabs had higher levels of four cryptocyanin forms. Gills of Bizerte crabs had higher levels of ferritin, three vitellogenins forms, lectin 4C, actin, and collagenolytic serine protease. Proteins with altered expression in crabs from Tunisia littoral are related to molting, oxidative stress and inflammation, innate immune response, and proteolysis.

iTRAQ analysis of hepatic proteins in free-living Mus spretus mice to assess the contamination status of areas surrounding Doñana National Park (SW Spain).

This work aims to develop and integrate new -omics tools that would be applicable to different ecosystem types for a technological updating of environmental evaluations. We used a 2nd-generation (iTRAQ-8plex) proteomic approach to identify/quantify proteins differentially expressed in the liver of free-living Mus spretus mice from Doñana National Park or its proximities. Mass spectrometry was performed in an LTQ Orbitrap system for iTRAQ reporter ion quantitation and protein identification using a Mus musculus database as reference. A prior IEF step improved the separation of the complex peptide mixture. Over 2000 identified proteins were altered, of which 118 changed by ≥2.5-fold in mice from at least two problem sites. Part of the results obtained with the iTRAQ analysis was confirmed by Western blot. Over 75% of the 118 proteins were upregulated in animals captured at polluted sites and only 16 proteins were downregulated. Upregulated proteins were involved in stress response; cell proliferation and apoptosis; signal transduction; metastasis or tumour suppression; xenobiotic export or vesicular trafficking; and metabolism. The downregulated proteins, all potentially harmful, were classified as oncoproteins and proteins favouring genome instability. The iTRAQ results presented here demonstrated that the survival of hepatic cells is compromised in animals living at polluted sites, which showed deep alterations in metabolism and the signalling pathways. The identified proteins may be useful as biomarkers of environmental pollution and provide insight about the metabolic pathways and/or physiological processes affected by pollutants in DNP and its surrounding areas.