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In the last years, "omics" approaches have been applied to study the toxicity of nanomaterials (NM) with the aim of obtaining insightful information on their biological effects. One of the most developed "omics" field, transcriptomics, expects to find unique profiles of differentially-expressed genes after exposure to NM that, besides providing evidence of their mechanistic mode of action, may also be used as biomarkers for biomonitoring purposes. Moreover, several NM have been associated with epigenetic alterations, i.e., changes in the regulation of gene expression caused by differential DNA methylation, histone tail modification and microRNA expression. Epigenomics research focusing on DNA methylation is increasingly common and the role of microRNAs is being better understood, either promoting or suppressing biological pathways. Moreover, the proteome is a highly dynamic system that changes constantly in response to a stimulus. Therefore, proteomics can identify changes in protein abundance and/or variability that lead to a better understanding of the underlying mechanisms of action of NM while discovering biomarkers. As to genomics, it is still not well developed in nanotoxicology. Nevertheless, the individual susceptibility to NM mediated by constitutive or acquired genomic variants represents an important component in understanding the variations in the biological response to NM exposure and, consequently, a key factor to evaluate possible adverse effects in exposed individuals. By elucidating the molecular changes that are involved NM toxicity, the new "omics" studies are expected to contribute to exclude or reduce the handling of hazardous NM in the workplace and support the implementation of regulation to protect human health.

In the last years, “omics” approaches have been applied to study the toxicity of nanomaterials (NM) with the aim of obtaining insightful information on their biological effects. One of the most developed “omics” field, transcriptomics, expects to find unique profiles of differentially expressed genes after exposure to NM that, besides providing evidence of their mechanistic mode of action, may also be used as biomarkers for biomonitoring purposes. Moreover, several NM have been associated with epigenetic alterations, i.e., changes in the regulation of gene expression caused by differential DNA methylation, histone tail modification and microRNA expression. Epigenomics research focusing on DNA methylation is increasingly common and the role of microRNAs is being better understood, either promoting or suppressing biological pathways. Moreover, the proteome is a highly dynamic system that changes constantly in response to a stimulus. Therefore, proteomics can identify changes in protein abundance and/ or variability that lead to a better understanding of the underlying mechanisms of action of NM while discovering biomarkers. As to genomics, it is still not well developed in nanotoxicology. Nevertheless, the individual susceptibility to NM mediated by constitutive or acquired genomic variants represents an important component in understanding the variations in the biological response to NM exposure and, consequently, a key factor to evaluate possible adverse effects in exposed individuals. By elucidating the molecular changes that are involved NM toxicity, the new “omics” studies are expected to contribute to exclude or reduce the handling of hazardous NM in the workplace and support the implementation of regulation to protect human health. This work was funded by FCT/ MCTES, project PTDC/SAU-PUB/32587/2017 and project PTDC/BTM-TEC/28554/2017 through national funds (PIDDAC), ToxOmics (UIDB/00009/2020; UIDP/00009/2020) and BioISI—Biosystems & Integrative Sciences Institute, Faculty of Sciences University of Lisbon, Lisbon, Portugal. It is also a result of the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by FCT, and the National Mass Spectrometry Network (RNEM)-FCT Infrastructure Program, Portugal. info:eu-repo/semantics/publishedVersion

Environmental tobacco smoke (ETS) has been recognized as a major health hazard by environmental and public health authorities worldwide. In Portugal, smoke-free laws are in force for some years, banning smoking in most indoor public spaces. However, in hospitality venues such as restaurants and bars, owners can still choose between a total smoke-free policy or a partial smoking restriction with designated smoking areas, if adequate reinforced ventilation systems are implemented. Despite that, a previous study showed that workers remained continuously exposed to higher ETS pollution in Lisbon restaurants and bars where smoking was still allowed, comparatively to total smoke-free venues. This was assessed by measurements of indoor PM2.5 and urinary cotinine, a biomarkers of tobacco smoke exposure, demonstrating that partial smoking restrictions do not effectively protect workers from ETS. The aim of the present work was to characterize effect and susceptibility biomarkers in non-smokers from those hospitality venues occupationally exposed to ETS comparatively to non-exposed ones. A group of smokers was also included for comparison. The sister chromatid exchange (SCE), micronucleus (MN) and comet assays in whole peripheral blood lymphocytes (PBLs) and the micronucleus assay in exfoliated buccal cells, were used as biomarkers of genotoxicity. Furthermore, a comet assay after ex vivo challenge of leukocytes with an alkylating agent, ethyl methanesulfonate (EMS), was used to analyze the repair capacity of those cells. Genetic polymorphisms in genes associated with metabolism and DNA repair were also included. The results showed no clear association between occupational exposure to ETS and the induction of genotoxicity. Interestingly, the leukocytes from non-smoking ETS-exposed individuals displayed lower DNA damage levels in response to the ex vivo EMS challenge, in comparison to those from non-exposed workers, suggesting a possible adaptive response. The contribution of individual susceptibility to the effect biomarkers studied was unclear, deserving further investigation. This work was funded by the Portuguese institutions Fundação Calouste Gulbenkian, Administração Central do Sistema de Saúde (ACSS) and FCT/MCTES through funding to ToxOmics (UIDB/00009/2020; UIDP/00009/2020). info:eu-repo/semantics/publishedVersion

In this study, we examined the effect of six months of positive airway pressure (PAP) therapy on Obstructive Sleep Apnea (OSA) red blood cell (RBC) proteome by two dimensional difference gel electrophoresis (2D-DIGE) - based proteomics followed by Western blotting (WB) validation. The discovered dysregulated proteins/proteoforms are associated with cell death, H2O2 catabolic/metabolic process, stress response, and protein oligomerization. Validation by nonreducing WB was performed for peroxiredoxin-2 (PRDX2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by using antibodies against the sulfinylated/sulfonylated cysteine of these proteins to better evaluate their redox&ndash, oligomeric states under OSA and/or in response to PAP therapy. The results indicated that the redox&ndash, oligomeric state of GAPDH and PRDX2 involving overoxidation by sulfinic/sulfonic acids were differentially modulated in OSA RBC, which might be compromising RBC homeostasis. PAP therapy by restoring this modulation induced a higher oligomerization of overoxidized GAPDH and PRDX2 in some patients that could be associated with eryptosis and the chaperone &ldquo, gain&rdquo, of function, respectively. This varied response following PAP may result from the complex interplay between OSA and OSA metabolic comorbidity. Hence, information on the redox status of PRDX2 and GAPDH in RBC will help to better recognize OSA subtypes and predict the therapeutic response in these patients. GAPDH monomer combined with body mass index (BMI) and PRDX2 S-S dimer combined with homeostatic model assessment for insulin resistance (HOMA-IR) showed to be very promising biomarkers to predict OSA and OSA severity, respectively.

© 2020 The Author(s)., [Introduction]: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. [Areas covered]: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. [Expert commentary]: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine., This paper was funded by the European cooperation in science and technology - COST action No. CA16113 - CliniMARK: ‘good biomarker practice’ to increase the number of clinically validated biomarkers.

2017 The Author. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)., This article presents proteomics data referenced in [1] Using proteomics-based evaluation of red blood cells (RBCs), we have identified differentially abundant proteins associated with Obstructive Sleep Apnea Syndrome (OSA). RBCs were collected from peripheral blood of patients with moderate/severe OSA or snoring at pre- (evening) and post-night (morning) polysomnography, so that proteome variations between these time points could be assessed. RBC cytoplasmic fraction depleted of hemoglobin, using Hemovoid(™) system, were analyzed by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), the 2D image software-based analyzed and relevant differentially abundant proteins identified by mass spectrometry (MS). MS identified 31 protein spots differentially abundant corresponding to 21 unique proteins possibly due to the existence of post-translational modification regulations. Functional analysis by bioinformatics tools indicated that most proteins are associated with catalytic, oxidoreductase, peroxidase, hydrolase, ATPase and anti-oxidant activity. At morning a larger numbers of differential proteins including response to chemical stimulus, oxidation reduction, regulation of catalytic activity and response to stress were observed in OSA. The data might support further research in OSA biomarker discovery and validation., Project partially supported by Harvard Medical School-Portugal Program (HMSP-ICJ/ 0022/2011), ToxOmics - Centre for Toxicogenomics and Human Health (FCT-UID/BIM/00009/2013), FCT/Poly-Annual Funding Program and FEDER/Saúde XXI Program (Portugal) and postdoctoral fellowship SFRH/BPD/43365/2008 of Fundação para a Ciência e a Tecnologia (FCT), Portugal.

© 2016 Published by Elsevier B.V., We have examined the effects of Obstructive Sleep Apnea (OSA) on red blood cell (RBC) proteome variation at evening/morning day time to uncover new insights into OSA-induced RBC dysfunction that may lead to OSA manifestations. Dysregulated proteins mainly fall in the group of catalytic enzymes, stress response and redox regulators such as peroxiredoxin 2 (PRDX2). Validation assays confirmed that at morning the monomeric/dimeric forms of PRDX2 were more overoxidized in OSA RBC compared to evening samples. Six month of positive airway pressure (PAP) treatment decreased this overoxidation and generated multimeric overoxidized forms associated with chaperone/transduction signaling activity of PRDX2. Morning levels of overoxidized PRDX2 correlated with polysomnographic (PSG)-arousal index and metabolic parameters whereas the evening level of disulfide-linked dimer (associated with peroxidase activity of PRDX2) correlated with PSG parameters. After treatment, morning overoxidized multimer of PRDX2 negatively correlated with fasting glucose and dopamine levels. Overall, these data point toward severe oxidative stress and altered antioxidant homeostasis in OSA RBC occurring mainly at morning time but with consequences till evening. The beneficial effect of PAP involves modulation of the redox/oligomeric state of PRDX2, whose mechanism and associated chaperone/transduction signaling functions deserves further investigation. RBC PRDX2 is a promising candidate biomarker for OSA severity and treatment monitoring, warranting further investigation and validation., Project partially supported by Harvard Medical School-Portugal Program (HMSPICJ/0022/2011), ToxOmics - Centre for Toxicogenomics and Human Health (FCT-UID/BIM/00009/2013), FCT/Poly-Annual Funding Program and FEDER/Saúde XXI Program (Portugal) and postdoctoral fellowship (SFRH/BPD/43365/2008) of Fundação para a Ciência e a Tecnologia (FCT), Portugal.

In this special issue, an updated overview on ‘where we are and where we are going in the area of top-down proteomics’ is given by Fornelli et al., one of the pioneering groups in top-down proteomics led by the proponent head of the term ‘proteoform’ and founder of the international Consortium in Top-Down Proteomics (CTDP), Neil Kelleher. Although being still technologically challenging, the rapid advances over the past years have demonstrated that the identification and characterization of thousands of proteoforms in high-throughput mode is today feasible for intact proteins< 30 kDa under denaturing condition. To further advance, the development of a new generation of instruments and softwares is still necessary, which may happen in the next coming years as predicted by the authors. The high molecular weight capability of native-top down proteomics, used today to characterize purified protein complexes under non-denaturing condition, is one of these technologies that could be innovated for high throughput top-down purposes. The main driver for this breakthrough is undoubtedly the wider acceptance and application of top-down approaches by proteomic researchers. This journey is already under way, as evidenced by the different articles presented in this special issue. info:eu-repo/semantics/publishedVersion

The quest to understand biological systems requires further attention of the scientific community to the challenges faced in proteomics. In fact the complexity of the proteome reaches uncountable orders of magnitudes. This means that significant technical and data-analytic innovations will be needed for the full understanding of biology. Current state of art mass spectrometry (MS) is probably our best choice for studying protein complexity and exploring new ways to use MS and MS derived data should be given higher priority. We present here a brief overview of visualization and statistical analyzes strategies for quantitative peptide values on an individual protein basis. These analysis strategies can help pinpoint protein modifications, splice and genomic variants of biological relevance. We demonstrated the application of these data analysis strategies using a bottom-up proteomics data set obtained in a drug profiling experiment. Furthermore, we have also observed that the presented methods are useful for studying peptide distributions from clinical proteomics samples from a large number of individuals. We expect that the presented data analysis strategy will be useful in the future to define functional protein variants in biological model systems and disease studies. Therefore robust software implementing these strategies is urgently needed. R.M. is supported by EXPL/DTP-PIC/0616/2013 Fundação para a Ciência e a Tecnologia (FCT) and FCT investigator program 2012. A.S.C. is supported by post-doctoral grant reference SFRH / BPD / 85569 / 2012 funded by FCT.

© The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved., α-Synuclein misfolding and aggregation is a hallmark in Parkinson's disease and in several other neurodegenerative diseases known as synucleinopathies. The toxic properties of α-synuclein are conserved from yeast to man, but the precise underpinnings of the cellular pathologies associated are still elusive, complicating the development of effective therapeutic strategies. Combining molecular genetics with target-based approaches, we established that glycation, an unavoidable age-associated post-translational modification, enhanced α-synuclein toxicity in vitro and in vivo, in Drosophila and in mice. Glycation affected primarily the N-terminal region of α-synuclein, reducing membrane binding, impaired the clearance of α-synuclein, and promoted the accumulation of toxic oligomers that impaired neuronal synaptic transmission. Strikingly, using glycation inhibitors, we demonstrated that normal clearance of α-synuclein was re-established, aggregation was reduced, and motor phenotypes in Drosophila were alleviated. Altogether, our study demonstrates glycation constitutes a novel drug target that can be explored in synucleinopathies as well as in other neurodegenerative conditions, Authors were supported by: H.V.M. (Fundação para a Ciência e Tecnologia (FCT), Portugal SFRH/BPD/64702/ 2009 and SFRH/BPD/109347/2015; EU FP7 project MEFOPA), L.M.A.O (FCT - SFRH/BD/23604/2005; CIRM-BMFB joint grant, 315050 AZ0101-31P6855), R.M.O. and T.S. (FCT SFRH/BPD/41416/2007; SFRH/ BPD/31209/2006); W.X. (Deutsche Forschungsgemeinschaft, SFB539/A3); C.B. and F.G. (Parkinson’s UK and the Medical Research Council, UK). S.E. is supported by Israel Academy of Sciences, Rappaport Family Institute for Research in the Medical Sciences, The Allen and Jewel Prince Center for Neurodegenerative Disorders of the Brain. T.F.O. (EMBO Installation Grant; Marie Curie IRG, Neurofold; DFG Center for Nanoscale Microscopy and Molecular Physiology of the Brain; I.C.M. (FCT SFRH/BPD/74287/2010; Investigador FCT IF/00772/ 2013). This work was supported by: FCT PTDC/SAUNEU/105215/2008, PTDC/QUI/73430/2006, PTDC/SAUENB/117013/2010, PTDC/NEU-OSD/5644/2014; EU FP7 project MEFOPA; CIRM-BMFB joint grant (315050 AZ0101-31P6855); Max Planck Society; and European Union (NEURASYNC PITNGA-2009-238316).

© 2017 Sociedade Portuguesa de Pneumologia. Published by Elsevier España, S.L.U. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)., Obstructive sleep apnea syndrome (OSAS) is a systemic inflammatory disease associated with cardiovascular consequences. Red blood cell distribution width (RDW), mean platelet volume (MPV), and platelet distribution width (PDW) are recognized biomarkers of cardiovascular morbidity/mortality. Limited data is available on the association between these parameters and OSAS severity and the relationship with positive airway pressure therapy (PAP). In this prospective study of male OSAS patients we analyzed hematological data in order to evaluate their value in predicting OSAS severity, the relationship with sleep parameters, and their behavior under PAP. Seventy-three patients were included (mean age 46.5 years), of which 36 were mild (49.3%), 10 moderate (13.7%), and 27 severe (37%). The mean RDW increased significantly with OSAS severity and showed a positive correlation with respiratory disturbance index and hypoxemic burdens. Additionally, a group of 48 patients (mean age 47.2 years) were submitted to PAP. After six months, red blood cell count, hemoglobin, hematocrit, and platelet count showed a significant decrease (p, Open Access funded by Sociedade Portuguesa de Pneumologia under a Creative Commons license

The field of clinical proteomics is faced with multiple challenges which need to be overcome in order to improve our understanding of human diseases and provide management solutions. Researchers interested in clinical proteomics assembled for a roundtable discussion at the European Association for Proteomics (EuPA) conference held in Glasgow in July 2012, to discuss these challenges and highlight the key areas for successful clinical proteomic studies. This report shares topics of discussion and the resulting stretch goals of clinical proteomics for researchers to strive towards.

In a previous study, evidence was provided that indoor secondhand tobacco smoke (SHS) air pollution remains high in Lisbon restaurants where smoking is allowed, regardless of the protective measures used. The aim of this study was to determine in these locations the levels of polycyclic aromatic hydrocarbons (PAH) associated with the particulate phase of SHS (PPAH), a fraction that contains recognized carginogens, such as benzo[a]pyrene (BaP). Data showed that restaurant smoking areas might contain PPAH levels as high as 110 ng/m(3), a value significantly higher than that estimated for nonsmoking areas (30 ng/m(3)) or smoke-free restaurants (22 ng/m(3)). The effective exposure to SHS components in restaurant smoking rooms was confirmed as cotinine levels found in workers' urine. Considering that all workers exhibited normal lung function, eventual molecular changes in blood that might be associated with occupational exposure to SHS and SHS-associated PPAH were investigated by measurement of two oxidative markers, total antioxidant status (TAS) and 8-hydroxyguanosine (8-OHdG) in plasma and serum, respectively. SHS-exposed workers exhibited higher mean levels of serum 8-OHdG than nonexposed workers, regardless of smoking status. By using a proteomics approach based on 2D-DIGE-MS, it was possible to identify nine differentially expressed proteins in the plasma of SHS-exposed nonsmoker workers. Two acute-phase inflammation proteins, ceruloplasmin and inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), were predominant. These two proteins presented a high number of isoforms modulated by SHS exposure with the high-molecular-weight (high-MW) isoforms decreased in abundance while low-MW isoforms were increased in abundance. Whether these expression profiles are due to (1) a specific proteolytic cleavage, (2) a change on protein stability, or (3) alterations on post-translational modification pattern of these proteins remains to be investigated. Considering that these events seem to precede the first symptoms of tobacco-related diseases, our findings might contribute to elucidation of early SHS-induced pathogenic mechanisms and constitute a useful tool for monitoring the effects of SHS on occupationally exposed individuals such as those working in the hospitality industry. Fundação Calouste Gulbenkian and Administração Central do Sistema de Saúde in Portugal have funded this work.

Uncovering unknown pathological mechanisms and body response to applied medication are the driving forces toward personalized medicine. In this post-genomic era, all eyes are turned to the proteomics field, searching for answers and explanations by investigating the gene end point functional units-proteins and their proteoforms. The development of cutting-edge mass spectrometric technologies and bioinformatics tools have allowed the life-science community to discover disease-specific proteins as biomarkers, which are often concealed by high sample complexity and dynamic range of abundance. Currently, there are several proteomics-based approaches to investigate the proteome. This chapter focuses on gold standard proteomics strategies and related issues toward candidate biomarker discovery, which may have diagnostic/prognostic as well as mechanistic utility in cancer drug resistance.

Structural and metabolic alterations in erythrocytes play an important role in the pathophysiology of Chronic Obstructive Pulmonary Disease (COPD). Whether these dysfunctions are related to the modulation of erythrocyte membrane proteins in patients diagnosed with COPD remains to be determined. Herein, a comparative proteomic profiling of the erythrocyte membrane fraction isolated from peripheral blood of smokers diagnosed with COPD and smokers with no COPD was performed using differential (16)O/(18)O stable isotope labeling. A total of 219 proteins were quantified as being significantly differentially expressed within the erythrocyte membrane proteomes of smokers with COPD and healthy smokers. Functional pathway analysis showed that the most enriched biofunctions were related to cell-to-cell signaling and interaction, hematological system development, immune response, oxidative stress and cytoskeleton. Chorein (VPS13A), a cytoskeleton related protein whose defects had been associated with the presence of cell membrane deformation of circulating erythrocytes was found to be down-regulated in the membrane fraction of erythrocytes obtained from COPD patients. Methemoglobin reductase (CYB5R3) was also found to be underexpressed in these cells, suggesting that COPD patients may be at higher risk for developing methemoglobinemia. This article is part of a Special Issue entitled: Integrated omics.

Environmental tobacco smoke (ETS), also referred to as secondhand smoke (SHS), is a major threat to public health and is increasingly recognized as an occupational hazard to workers in the hospitality industry. Therefore, several countries have implemented smoke-free regulations at hospitality industry sites. In Portugal, since 2008, legislation partially banned smoking in restaurants and bars but until now no data have been made available on levels of indoor ETS pollution/exposure at these locations. The aim of this study was to examine the occupational exposure to ETS/SHS in several restaurants in Lisbon, measured by indoor fine particles (PM(2.5)) and urinary cotinine concentration in workers, after the partial smoking ban in Portugal. Results showed that the PM(2.5) median level in smoking designated areas was 253 μg/m³, eightfold higher than levels recorded in canteens or outdoor. The nonsmoking rooms of mixed restaurants exhibited PM(2.5) median level of 88 μg/m³, which is higher than all smoke-free locations studied, approximately threefold greater than those found in canteens. Importantly, urinary cotinine concentrations were significantly higher in nonsmoker employees working in those smoking designated areas, confirming exposure to ETS. The proportion of smokers in those rooms was found to be significantly positively correlated with nonsmoker urinary cotinine and indoor PM(2.5) levels, establishing that both markers were occupational-ETS derived. The use of reinforced ventilation systems seemed not to be sufficient to decrease the observed ETS pollution/exposure in those smoking locations. Taken together, these findings demonstrate that the partial restrictions on smoking in Portuguese venues failed to provide adequate protection to their employees, irrespective of protective measures used. Therefore, a smoke-free legislation protecting individuals from exposure to ETS/SHS in all public places and workplaces is urgently needed in Portugal.

Portuguese legislation prohibits smoking in most indoor public spaces. However, some restaurants/bars remain, in which smoking is still allowed, representing a potential risk for employees, particularly for chronic respiratory diseases and cancer development. The objectives of this project were to compare the indoor air quality of some restaurants with and without smoking permission and, in their workers, to search for associations between respiratory dysfunctions and biomarkers of exposure, biological effects (DNA and proteome alterations) and susceptibility. Herein, we present data on DNA damage and genetic susceptibility in 37 workers occupationally exposed to environmental tobacco smoke (ETS) and from 44 non-exposed workers. DNA damage was assessed by the comet assay in blood leukocytes and by micronucleus (MN) analysis in buccal cells. DNA repair capacity was evaluated by a challenge of blood cells with EMS (32 μM), preceding the comet assay. Polymorphisms in metabolism (GSTP1, GSTM and GSTT) and DNA-repair genes (hOGG1,XRCC1, XRCC3, and NBS1) were analysed by PCR/RFLP. No significant differences in the MN frequency and in level of DNA damage was observed between ETS and non-ETS groups. However, challenge with EMS resulted in a significantly lower level of DNA breaks in ETS-exposed as compared to non-exposed workers (P

A comprehensive proteomic profiling of nasal epithelium (NE) is described. This study relies on simple subcellular fractionation used to obtain soluble- and membrane-enriched fractions followed by 2-dimensional liquid chromatography (2D-LC) separation and tandem mass spectrometry (MS/MS). The cells were collected using a brushing technique applied on NE of clinically evaluated volunteers. Subsequently, the soluble- and the membrane-protein enriched fractions were prepared and analyzed in parallel using 2D-LC-MS/MS. In a set of 1482 identified proteins, 947 (63.9%) proteins were found to be associated to membrane fraction. Grand average hydropathy value index (GRAVY) analysis, the transmembrane protein mapping and annotations of primary location deposited in the Human Protein Reference Database (HPRD) confirmed an enrichment of hydrophobic proteins on this dataset. Ingenuity Pathway Analysis (IPA) of soluble fraction revealed an enrichment of molecular and cellular functions associated with cell death, protein folding and drug metabolism while in membrane fraction showed an enrichment of functions associated with molecular transport, protein trafficking and cell-to-cell signaling and interaction. The IPA showed similar enrichment of functions associated with cellular growth and proliferation in both soluble and membrane subproteomes. This finding was in agreement with protein content analysis using exponentially modified protein abundance index (emPAI). A comparison of our data with previously published studies focusing on respiratory tract epithelium revealed similarities related to identification of proteins associated with physical barrier function and immunological defence. In summary, we extended the NE molecular profile by identifying and characterizing proteins associated to pivotal functions of a respiratory epithelium, including the control of fluid volume and ionic composition at the airways' surface, physical barrier maintenance, detoxification and immunological defence. The extent of similarities supports the applicability of a less invasive analysis of NE to assess prognosis and treatment response of lung diseases such as asthma, cystic fibrosis and chronic obstructive pulmonary disease., Graphical abstract This work represents the most complete proteome characterization of nasal epithelium (NE) describing its relevant functions. NE usefulness in lung biomedical research is highlighted based on observed similarities with bronchial epithelial proteome. Research highlights ► Sub-fractionation of nasal epithelial cells (NEC) to improve the detection of low abundance proteins. ► Global proteome characterization based on a gel-free SCX-RPLC- chromatography coupled with MS/MS approach. ► Characterization of cell membrane proteins using a methanol-based solubilization before tryptic digestion. ► Pathway Analysis to profile molecular and cellular functions overrepresented in nasal epithelium. ► Extensive comparison of our data with previously published studies focusing on respiratory tract epithelium.

Complementary 2D-PAGE and ‘shotgun’ LC–MS/MS approaches were combined to identify medium and low-abundant proteins in sera of Cystic Fibrosis (CF) patients (mild or severe pulmonary disease) in comparison with healthy CF-carrier and non-CF carrier individuals aiming to gain deeper insights into the pathogenesis of this multifactorial genetic disease. 78 differentially expressed spots were identified from 2D-PAGE proteome profiling yielding 28 identifications and postulating the existence of post-translation modifications (PTM). The ‘shotgun’ approach highlighted altered levels of proteins actively involved in CF: abnormal tissue/airway remodeling, protease/antiprotease imbalance, innate immune dysfunction, chronic inflammation, nutritional imbalance and Pseudomonas aeruginosa colonization. Members of the apolipoproteins family (VDBP, ApoA-I, and ApoB) presented gradually lower expression from non-CF to CF-carrier individuals and from those to CF patients, results validated by an independent assay. The multifunctional enzyme NDKB was identified only in the CF group and independently validated by WB. Its functions account for ion sensor in epithelial cells, pancreatic secretion, neutrophil-mediated inflammation and energy production, highlighting its physiological significance in the context of CF. Complementary proteomics-based approaches are reliable tools to reveal pathways and circulating proteins actively involved in a heterogeneous disease such as CF.

The achievement and maintenance of a protein native conformation is a very complex cellular process involving a multitude of key factors whose contribution to a successful folding remains to be elucidated. On top of this, it is known that correct folding is crucial for proteins to play their normal role and, consequently, for the maintenance of cellular homeostasis or proteostasis. If the folding process is affected, the protein is unable to achieve its native conformation, compromising its life and function, and a pathological condition may arise. Protein-misfolding diseases are characterized by either formation of protein aggregates that are toxic to the cell (gain-of-toxic-function diseases) or by an incorrect processing of proteins, which leads to a deficiency in protein activity (loss-of-function diseases). In this article we have focused on proteomics advances in the molecular knowledge of protein-misfolding diseases with direct impact on possible key players in F508del-CFTR processing and trafficking.

F508del-CFTR, the most common mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, disrupts intracellular trafficking leading to cystic fibrosis (CF). The trafficking defect of F508del-CFTR can be rescued by simultaneous inactivation of its four RXR motifs (4RK). Proteins involved in the F508del-CFTR trafficking defect and/or rescue are therefore potential CF therapeutic targets. We sought to identify these proteins by investigating differential proteome modulation in BHK cells over-expressing wt-CFTR, F508del-CFTR or the revertant F508del/4RK-CFTR. By 2-dimensional electrophoresis-based proteomics and western blot approaches we demonstrated that over-expression of F508del/4RK-CFTR modulates the expression of a large number of proteins, many of which are reported interactors of CFTR and/or 14-3-3 with potential roles in CFTR trafficking. GRP78/BiP, a marker of ER stress and unfolded protein response (UPR), is up-regulated in cells over-expressing either F508del-CFTR or F598del/4RK-CFTR. However, over-expression of F508del/4RK-CFTR induces the up-regulation of many other UPR-associated proteins (e.g. GRP94, PDI, GRP75/mortalin) and, interestingly, the down-regulation of proteasome components associated with CFTR degradation, such as the proteasome activator PA28 (PSME2) and COP9 signalosome (COPS5/CSN5). Moreover, the F508del-CFTR-induced proteostasis imbalance, which involves some heat shock chaperones (e.g. HSP72/Hpa2), ER-EF-hand Ca2+-binding proteins (calumenin) and the proteasome activator PA28 (PSME2), tends to be ‘restored’, i.e., in BHK cells over-expressing F508del/4RK-CFTR those proteins tend to have expression levels similar to the wild-type ones. These findings indicate that a particular cellular environment orchestrated by the UPR contributes to and/or is compatible with F508del/4RK-CFTR rescue.

Objectives: The aim of this work was to establish protein profiles in serum and nasal epithelial cells of cystic fibrosis individuals in comparison with controls, asthma and chronic obstructive pulmonary disease patients for specific biomarker signatures identification. Design and methods: Protein extracts were analyzed by Surface Enhanced Laser Desorption/Ionization Time-Of-Flight Mass-Spectrometry (SELDI-TOF-MS). Results: The mass spectra revealed a set of peaks with differential expression in serum and nasal cells among the different groups studied, resulting into peak signatures representative/specific of each pathology. Logistic regressions were applied to those peaks; sensitivity, specificity, Youden's indexes and area under the curve (AUC) of the respective receiver operating characteristic (ROC) curves were compared. Discussion: Multivariate analysis demonstrated that combination of peaks has a better predictive value than the individual ones. These protein signatures may serve as diagnostic/prognostic markers for the studied diseases with common clinical features, or as follow-up assessment markers of therapeutic interventions.

To gain insight into the proteins potentially involved in the low temperature-induced F508del-CFTR rescue process, we have explored by two-dimensional electrophoresis (2DE) the proteome of BHK cell lines expressing wt or F508del-CFTR, grown at 37 degrees C or 26 degrees C/24h or 26 degrees C/48h followed by 3h of metabolic labelling with [(35)S]-methionine. A set of 139 protein spots (yielding 125 mass spectrometry identifications) was identified as differentially expressed (p ANOVA

Hepatitis delta virus (HDV) infects human hepatocytes already infected with the hepatitis B virus increasing about ten fold the risk of cirrhosis and fulminant hepatitis. The lack of an appropriate cell culture system capable of supporting virus replication has so far impaired the detailed investigation of the HDV biology including the identification of host factors involved in pathogenesis. Here, we made use of a HDV cDNA stably transfected cell line, Huh7-D12, in a proteomic approach to identify the changes in the protein expression profiles in human liver cells that arise as a consequence of HDV replication. Total protein extracts from Huh7-D12 cells and of the corresponding non transfected human liver carcinoma cell line, Huh7, were separated by 2-DE. Differentially expressed spots were identified by MALDI-TOF followed by database searching. We identified 23 differentially expressed proteins of which 15 were down regulated and 8 up regulated in Huh7-D12 cells. These proteins were found to be involved in different cellular pathways. The down regulation of the histone H1-binding protein and of triosephosphate isomerase was confirmed by Real time PCR, and the up regulation of the La protein and lamin A/C was validated by western blot.

Combined results from laser capture microdissection of mouse airway epithelial cells followed by high power (MALDI-FTICR) MS, and fluorescent two-dimensional gel elctrophoresis (2D-DIGE) of the whole lung, allowed us to identify proteins differentially expressed after naphthalene induced airway injury. Further, we discovered several novel aspects of Cystic Fibrosis (CF) lung pathology in an F508del-Cftr mouse model using this approach. The combined MALDI-FTICR-MS and 2D-DIGE data show that lung carbonyl reductase (CBR2), involved in prostaglandin metabolism, converting PGE2 to PGF2alpha, is localized to airway cells and is reduced 2-fold in mutant mice compared to normal, both before and after challenge. Further, we observe a downregulation of two key enzymes of retinoic acid metabolism after injury, which is more pronounced in CF mutant mice. These data show that state-of-the-art proteomics can be used to evaluate airway injury in small cell samples. Further, the results suggest the involvement of prostaglandin and retinoic acid metabolism in the abnormal responses of CF mutant mice to injury.

Hepatitis delta virus (HDV) infection of human hepatocytes infected with the hepatitis B virus (HBV) is associated with increased liver damage and risk of fulminant disease. Although considerable progress has been made towards the elucidation of the mechanisms of HDV replication and pathogenesis, little is still known about the host factors involved in the different steps of the replication cycle. Here, we made use of a proteomic approach to analyse the global alterations in protein expression that arise in human hepatocytes separately transfected with each of the HDV components. Huh7 cells were transiently transfected with plasmids that code for the small delta antigen (S-HDAg), large delta antigen (L-HDAg), genomic RNA (gRNA), and antigenomic RNA (agRNA), respectively. Total protein extracts were separated by 2-DE and differentially expressed spots were identified by MALDI-TOF followed by database searching. We identified 32 proteins known to be involved in different pathways namely nucleic acid metabolism, protein metabolism, transport, signal transduction, apoptosis, and cell growth. Moreover, the down regulation of hnRNP D, HSP105, and triosephosphate isomerase was further confirmed by Real time PCR.

Ceruloplasmin (CP) is a multicopper oxidase involved in the acute phase reaction to stress. Although the physiological role of CP is uncertain, its role in iron (Fe) homeostasis and protection against free radical-initiated cell injury has been widely documented. Previous studies showed the existence of two molecular isoforms of CP: secreted CP (sCP) and a membrane glycosylphosphatidylinositol (GPI)-anchored form of CP (GPI-CP). sCP is produced mainly by the liver and is abundant in human serum whereas GPI-CP is expressed in mammalian astrocytes, rat leptomeningeal cells, and Sertolli cells. Herein, we show using RT-PCR that human peripheral blood lymphocytes (huPBL) constitutively express the transcripts for both CP molecular isoforms previously reported. Also, expression of CP in huPBL is demonstrated by immunofluorescence with confocal microscopy and flow cytometry analysis using cells isolated from healthy blood donors with normal Fe status. Importantly, the results obtained show that natural killer cells have a significantly higher CP expression compared to all other major lymphocyte subsets. In this context, the involvement of lymphocyte-derived CP on host defense processes via its anti/prooxidant properties is proposed, giving further support for a close functional interaction between the immune system and the Fe metabolism.

The proteome project, initiated in 1995, was made possible by 2-DE combined with MS. The project main objective was and remains the identification of all proteins expressed by a cell, tissue or organism in a given time and condition. Following this objective, the global profiling of proteins in health versus pathological state by the 2-DE/MS-based proteomic approach has contributed to the elucidation of the basic mechanisms of disease by discovering candidate disease biomarkers and disease targets for new drug development. This review will briefly summarize the historical evolution of 2-DE up to today, and review 2-DE/MS technology and its specific methods of study of immunoresponse (immunoproteomics), PTM of proteins, complex protein-protein interactions (interactome), the proteome of cell membrane and intracellular proteome turnover in disease biomarker discovery.

Potential biological markers for cystic fibrosis (CF) lung disease were identified by comparative proteomics profiling of nasal cells from deletion of phenylalanine residue 508 (F508del)-homozygous CF patients and non-CF controls. From the non-CF 2-DE gels, 65 spots were identified by MS, and a reference 2-DE map was thus established. The majority of those correspond to ubiquitously expressed proteins. Consistent with the epithelial origin of this tissue, some of the identified proteins are epithelial markers (e.g. cytokeratins, palate lung and nasal epithelium clone protein (PLUNC), and squamous cell carcinoma antigen 1). Comparison of this protein profile with the one similarly obtained for CF nasal cells revealed a set of differentially expressed proteins. These included proteins related to chronic inflammation and some others involved in oxidative stress injury. Alterations were also observed in the levels of cytoskeleton proteins, being probably implicated with cytoskeleton organization changes described to occur in CF-airways. Lower levels were found for some mitochondrial proteins suggesting an altered mitochondrial metabolism in CF. Differential expression was also found for two more enzymes that have not been previously associated to CF. Further studies will clarify the involvement of such proteins in CF pathophysiology and whether they are targets for CF therapy.

Interest in the biochemical detection of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein followed soon after cloning of the gene and prediction of the protein structure. Ever since, antibodies (Abs) have been produced and used to detect CFTR in both heterologously and endogenously expressing cells and tissues. Although designed to be sensitive and specific, these Abs produce, in most cases, unsatisfactory results when used for the biochemical detection of CFTR either by Western blot or by immunoprecipitation. The lack of Abs that can reliably detect the CFTR protein is a major constraint to studies of CF. We compared 14 different Abs for their ability to detect CFTR in both stably transfected and endogenously expressing cell lines.

Numerous factors, other than mutations in the CFTR gene, affect the phenotypic variability of cystic fibrosis (CF). With a two-dimensional electrophoresis (2-DE) analysis of total protein expression profiles (proteomics) of CF versus non-CF cells it is possible to obtain an integrative picture of CF cellular alterations. Through this approach, proteins that interact differently with wild type- and mutant-CFTR can also be identified (interactomics). This can provide insight into CF pathophysiology as well as clues for novel therapeutic targets. Additionally, protein profiling can ultimately identify novel disease markers with the potential for a CF diagnosis not based on the analysis of CFTR gene.

A561E, a novel cystic fibrosis (CF) associated mutation in the first nucleotide binding domain of CFTR, is the second most common CF mutation in Portugal. Properties of the A561E-CFTR protein were studied by immunoblotting, pulse-chase, immunocytochemistry, and MQAE halide-efflux assay in stably transfected BHK cells. Altogether, results presented here suggest that A561E causes protein mislocalization in the endoplasmic reticulum where the mutant protein must be trapped by the quality control mechanism. We conclude that A561E originates a protein trafficking defect, thus belonging to class II of CFTR mutations. As it is the case for F508del-CFTR (the most common CF mutant), low temperature treatment partially rescues a functional A561E-CFTR channel, suggesting that substitution of glutamic acid for alanine at position 561 does not completely abolish CFTR function. Pharmacological strategies previously reported for treatment of CF patients with the F508del mutation could thus be also effective in CF patients bearing the A561E mutation.

During the first decade of this millennium, we have witnessed of the most remarkable advances in the field of proteomics since the word proteome was introduced by Marc Wilkins in 1995. High degree of technical innovations has been successfully achieved to meet the ongoing challenges mostly associated with the need to know and map all proteins, their variants and functional networks in a given cell/tissue/organism at a particular time/condition. The best example of this ambition is the Human Proteome Project (HPP), launched by the Human Proteome Organization (HUPO), designed to map the entire human proteome. Completion of this project will certainly enhance our understanding of human cell/molecular biology and provide new tools for better health and disease control

We investigated the molecular effects of glucosamine supplements, a popular and safe alternative to nonsteroidal anti-inflammatory drugs, for decreasing pain, inflammation, and maintaining healthy joints. Numerous studies have reported an array of molecular effects after glucosamine treatment. We questioned whether the differences in the effects observed in previous studies were associated with the focus on a specific subproteome or with the use of specific cell lines or tissues. To address this question, global mass spectrometry- and transcription array-based glucosamine drug profiling was performed on malignant cell lines from different stages of lymphocyte development. We combined global label-free MS-based protein quantitation with an open search for modifications to obtain the best possible proteome coverage. Our data were largely consistent with previous studies in a variety of cellular models. We mainly observed glucosamine induced O-GlcNAcylation/O-GalNAcylation (O-HexNAcylation); however, we also observed global and local changes in acetylation, methylation, and phosphorylation. For example, our data provides two additional examples of "yin-yang" between phosphorylation and O-HexNAcylation. Furthermore, we mapped novel O-HexNAc sites on GLU2B and calnexin. GLU2B and calnexin are known to be located in the endoplasmic reticulum (ER) and involved in protein folding and quality control. The O-HexNAc sites were regulated by glucosamine treatment and correlated with the up-regulation of the ER stress marker GRP78. The occupancy of O-HexNAc on GLU2B and calnexin sites differed between the cytosolic and nuclear fractions with a higher occupancy in the cytosolic fraction. Based on our data we propose the hypothesis that O-HexNAc either inactivates calnexin and/or targets it to the cytosolic fraction. Further, we hypothesize that O-HexNAcylation induced by glucosamine treatment enhances protein trafficking.

Obstructive sleep apnea (OSA) is an underdiagnosed common public health concern causing deleterious effects on metabolic and cardiovascular health. Although much has been learned regarding the pathophysiology and consequences of OSA in the past decades, the molecular mechanisms associated with such processes remain poorly defined. The advanced high-throughput proteomics-based technologies have become a fundamental approach for identifying novel disease mediators as potential diagnostic and therapeutic targets for many diseases, including OSA. Here, we briefly review OSA pathophysiology and the technological advances in proteomics and the first results of its application to address critical issues in the OSA field. Work partially supported by Harvard Medical School – Portugal Program (HMSP-ICJ/0022/2011), Fundação para a Ciência e a Tecnologia (FCT)/Poly-Annual Funding Program and FEDER/Saúde XXI Program (Portugal) and postdoctoral FCT-fellowship (SFRH/BPD/43365/2008).

We characterized the 3272-26A-->G mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, creating an alternative acceptor splice site in intron 17a, that competes with the normal one, although we predict from consensus values, with lower efficiency. We analyzed five Cystic Fibrosis (CF) Portuguese patients with the 3272-26A-->G/F508del genotype. Besides clinical and haplotype characterization of those patients, we report here results from CFTR transcript analysis in nasal brushings from all five patients. RT-PCR analysis supports alternative splicing in all patients and carriers, but not in controls. By sequencing, we determined that the alternative transcript includes 25 nucleotides from intron 17a, which predictively cause frameshift and a premature stop codon. The use of this alternative splice site causes a reduction in the levels of normal transcripts from the allele with this mutation and, most probably, of normal protein as well. By immunocytochemistry of both epithelial primary cell cultures and slices from CF polyps, CFTR protein is detected at the cell membrane, with three different antibodies. Ussing chamber analysis of one nasal polyp shows a high sodium absorption, characteristic of CF. Altogether, the results suggest that the main defect caused by the 3272-26A-->G mutation is a reduction in normal CFTR transcripts and protein and therefore this mutation should be included in class V, according to Zielenski and Tsui.

Woody plants are particularly difficult to investigate due to high phenolic, resin, and tannin contents and laborious sample preparation. In particular, protein isolation from woody plants for two-dimensional gel electrophoresis (2-DE) is challenging as secondary metabolites negatively interfere with protein extraction and separation. In this study, three protein extraction protocols, using TCA, phenol and ethanol as precipitation or extraction agents, were tested in order to select the more efficient for woody recalcitrant plant gel-based proteomics. Grapevine leaves, pine needles and cork oak ectomycorrhizal roots were used to represent woody plant species and tissues. The phenol protocol produced higher quality 2-DE gels, with increased number of resolved spots, better spot focusing and representation of all molecular mass and isoelectric point ranges tested. In order to test the compatibility of the phenol extracted proteomes with protein identification several spots were excised from the phenol gels and analyzed by mass spectrometry (MALDI-TOF/TOF). Regardless the incomplete genome/protein databases for the plant species under analysis, 49 proteins were identified by Peptide Mass Fingerprint (PMF). Proteomic data have been deposited to the ProteomeXchange with identifier PXD000224. Our results demonstrate the complexity of protein extraction from woody plant tissues and the suitability of the phenol protocol for obtaining high quality protein extracts for efficient 2-DE separation and downstream applications such as protein identification by mass spectrometry. Electronic supplementary material The online version of this article (doi:10.1186/2193-1801-2-210) contains supplementary material, which is available to authorized users.

α-synuclein (α-syn) is the major component of Lewy bodies, a pathological hallmark of Parkinson's disease and other synucleinopathies. The characterization of α-syn post-translational modifications (PTMs), thought to interfere with its aggregation propensity and cellular signaling, has been limited by the availability of extraction methods of endogenous protein from cells and tissues, and by the availability of antibodies toward α-syn PTMs. Here, by taking advantage of α-syn thermostability, we applied a method to achieve high enrichment of soluble α-syn both from cultured cells and brain tissues followed by proteomics analysis. Using this approach, we obtained 98% α-syn sequence coverage in a variety of model systems, including a transgenic mouse model of PD, and validated the strategy by identifying previously described PTMs such as phosphorylation and N-terminal acetylation. Our findings demonstrate that this procedure overcomes existing technical limitations and can be used to facilitate the systematic study of α-syn PTMs, thereby enabling the clarification of their role under physiological and pathological conditions. Ultimately, this approach may enable the development of novel biomarkers and strategies for therapeutic intervention in synucleinopathies. In this study, we describe a method for enriching alpha-synuclein (α-syn) from a variety of biological samples, from cultured cells to brain tissues. Enrichment of α-syn was achieved by heating samples, further facilitating the identification of specific post-translational modifications by immunoblot, or mass spectrometry-based techniques. This approach will contribute to the clarification of the role of α-syn PTMs in Parkinson's disease.