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Understanding disease pathology often does not require an overall proteomic analysis of clinical samples but rather the analysis of different, often rare, subpopulations of cells in a heterogeneous mixture of cell types. For the isolation of pre-specified cellular subtypes, fluorescence activated cell sorting (FACS) is commonly used for its ability to isolate the required cell populations with high purity, even of scarce cell types. The proteomic analysis of a limited number of FACS-sorted cells, however, is very challenging as both sample preparation inefficiencies and limits in terms of instrument sensitivity are present. In this study, we used CD14+CD15+ immune cells sorted out of peripheral blood mononuclear cells isolated from whole blood to improve and evaluate FACS-based proteomics. To optimize both the protein extraction protocol and the mass spectrometry (MS) data acquisition method, PBMCs as well as commercialized HeLa digest were used. To reflect the limited number of sorted cells in some clinical samples, different numbers of sorted cells (1000, 5000, 10,000, or 50,000) were used. This allowed comparing protein profiles across samples with limited protein material and provided further insights in the benefits and limitations of using a very limited numbers of cells.
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Separação Celular/métodos , Citometria de Fluxo/métodos , Proteômica/métodos , Células HeLa , Humanos , Leucócitos/metabolismo , Leucócitos Mononucleares/metabolismo , Espectrometria de Massas/métodos , Proteoma/metabolismoRESUMO
Over the past few years, awareness has risen that for mass-spectrometry-based proteomics methods to mature into everyday analytical and clinical practices, extensive quality assessment is mandatory. A currently overlooked source of qualitative information originates from the mass spectrometer itself. Apart from the actual mass spectral data, raw-data objects also contain parameter settings and sensory information about the mass instrument. This information gives a detailed account of the operation of the instrument, which eventually can be related to observations in mass spectral data. The advantage of instrument information at the lowest level is the high sensitivity to detect emerging defects in a timely fashion. To this end, we introduce the Instrument MONitoring DataBase (iMonDB), which allows us to automatically extract, store, and manage the instrument parameters from raw-data objects into a highly efficient database structure. This enables us to monitor the instrument parameters over a considerable time period. Time course information about the instrument performance is necessary to define the normal range of operation and to detect anomalies that may correlate with instrument failure. The proposed tools foster an additional handle on quality control and are released as open source under the permissive Apache 2.0 license. The tools can be downloaded from https://bitbucket.org/proteinspector/imondb.
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Bases de Dados Factuais , Espectrometria de Massas/normas , Software , Análise de Falha de Equipamento , Humanos , Proteômica/instrumentação , Controle de QualidadeRESUMO
Urinary extracellular vesicles (EVs) are an attractive source of bladder cancer biomarkers. Here, a protein biomarker discovery study was performed on the protein content of small urinary EVs (sEVs) to identify possible biomarkers for the primary diagnosis and recurrence of non-muscle-invasive bladder cancer (NMIBC). The sEVs were isolated by ultrafiltration (UF) in combination with size-exclusion chromatography (SEC). The first part of the study compared healthy individuals with NMIBC patients with a primary diagnosis. The second part compared tumor-free patients with patients with a recurrent NMIBC diagnosis. The separated sEVs were in the size range of 40 to 200 nm. Based on manually curated high quality mass spectrometry (MS) data, the statistical analysis revealed 69 proteins that were differentially expressed in these sEV fractions of patients with a first bladder cancer tumor vs. an age- and gender-matched healthy control group. When the discriminating power between healthy individuals and first diagnosis patients is taken into account, the biomarkers with the most potential are MASP2, C3, A2M, CHMP2A and NHE-RF1. Additionally, two proteins (HBB and HBA1) were differentially expressed between bladder cancer patients with a recurrent diagnosis vs. tumor-free samples of bladder cancer patients, but their biological relevance is very limited.
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Ultrafiltração , Neoplasias da Bexiga Urinária , Humanos , Neoplasias da Bexiga Urinária/diagnóstico , Bexiga Urinária/metabolismo , Biomarcadores Tumorais/metabolismo , Cromatografia em GelRESUMO
INTRODUCTION: Antibody-mediated rejection (ABMR) impacts kidney allograft outcome. The diagnosis is made based on findings from invasive kidney transplant biopsy specimens. The aim of this study was to identify a noninvasive urinary protein biomarker for ABMR after kidney transplantation. METHODS: We performed a multicenter case-control study to identify a urinary biomarker for ABMR (training cohort, n = 249) and an independent, prospective multicenter cohort study for validation (n = 391). We used concomitant biopsies to classify the samples according to the Banff classification. After untargeted protein identification and quantification, we used a support vector machine to train the model in the training cohort. The primary endpoint was the diagnostic accuracy of the urinary biomarker for ABMR in the validation cohort. RESULTS: We identified a set of 10 urinary proteins that accurately discriminated patients with (n = 60) and without (n = 189) ABMR in the training cohort with an area under the curve (AUC) of 0.98 (95% confidence interval [CI], 0.96-1.00). The diagnostic accuracy was maintained in the validation cohort (AUC, 0.88; 95% CI, 0.8-0.93) for discriminating the presence (n = 43) from the absence (n = 348) of ABMR. The negative predictive value of the 10-protein marker set for exclusion of ABMR was 0.99, and the positive predictive value was 0.33. The diagnostic accuracy was independent of the reason for performing the biopsy, time after transplantation, and better than the accuracy of gross proteinuria (AUC, 0.76). CONCLUSIONS: We identified and validated a urinary protein biomarker set that can be used to exclude ABMR.
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The quality and relevance of nanosafety studies constitute major challenges to ensure their key role as a supporting tool in sustainable innovation, and subsequent competitive economic advantage. However, the number of apparently contradictory and inconclusive research results has increased in the past few years, indicating the need to introduce harmonized protocols and good practices in the nanosafety research community. Therefore, we aimed to evaluate if best-practice training and inter-laboratory comparison (ILC) of performance of the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay for the cytotoxicity assessment of nanomaterials among 15 European laboratories can improve quality in nanosafety testing. We used two well-described model nanoparticles, 40-nm carboxylated polystyrene (PS-COOH) and 50-nm amino-modified polystyrene (PS-NH2). We followed a tiered approach using well-developed standard operating procedures (SOPs) and sharing the same cells, serum and nanoparticles. We started with determination of the cell growth rate (tier 1), followed by a method transfer phase, in which all laboratories performed the first ILC on the MTS assay (tier 2). Based on the outcome of tier 2 and a survey of laboratory practices, specific training was organized, and the MTS assay SOP was refined. This led to largely improved intra- and inter-laboratory reproducibility in tier 3. In addition, we confirmed that PS-COOH and PS-NH2 are suitable negative and positive control nanoparticles, respectively, to evaluate impact of nanomaterials on cell viability using the MTS assay. Overall, we have demonstrated that the tiered process followed here, with the use of SOPs and representative control nanomaterials, is necessary and makes it possible to achieve good inter-laboratory reproducibility, and therefore high-quality nanotoxicological data.
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On average a human cell type expresses around 10,000 different protein coding genes synthesizing all the different molecular forms of the protein product (proteoforms) found in a cell. In a typical shotgun bottom up proteomic approach, the proteins are enzymatically cleaved, producing several 100,000â¯s of different peptides that are analyzed with liquid chromatography-tandem mass spectrometry (LC-MSMS). One of the major consequences of this high sample complexity is that coelution of peptides cannot be avoided. Moreover, low abundant peptides are difficult to identify as they have a lower chance of being selected for fragmentation due to ion-suppression effects and the semi-stochastic nature of the precursor selection in data-dependent shotgun proteomic analysis where peptides are selected for fragmentation analysis one-by-one as they elute from the column. In the current study we explore a simple novel approach that has the potential to counter some of the effect of coelution of peptides and improves the number of peptide identifications in a bottom-up proteomic analysis. In this method, peptides from a HeLa cell digest were eluted from the reverse phase column using three different elution solvents (acetonitrile, methanol and acetone) in three replicate reversed phase LC-MS/MS shotgun proteomic analysis. Results were compared with three technical replicates using the same solvent, which is common practice in proteomic analysis. In total, we see an increase of up to 10% in unique protein and up to 30% in unique peptide identifications from the combined analysis using different elution solvents when compared to the combined identifications from the three replicates of the same solvent. In addition, the overlap of unique peptide identifications common in all three LC-MS analyses in our approach is only 23% compared to 50% in the replicates using the same solvent. The method presented here thus provides an easy to implement method to significantly reduce the effects of coelution and ion suppression of peptides and improve protein coverage in shotgun proteomics. Data are available via ProteomeXchange with identifier PXD011908.
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Cromatografia Líquida/métodos , Proteoma/química , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Células HeLa , Humanos , Peptídeos/químicaRESUMO
Advanced non-small-cell lung cancer (NSCLC) is generally linked with a poor prognosis and is one of the leading causes of cancer-related deaths worldwide. Since only a minority of the patients respond well to chemotherapy and/or targeted therapies, immunotherapy might be a valid alternative in the lung cancer treatment field, as immunotherapy attempts to strengthen the body's own immune response to recognize and eliminate malignant tumor cells. However, positive response patterns to immunotherapy remain unclear. In this study, we demonstrate how immune-related factors could be visualized from single NSCLC tissue sections (Biobank@UZA) while retaining their spatial information by using matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI), in order to unravel the molecular profile of NSCLC patients. In this way, different regions in lung cancerous tissues could be discriminated based on the molecular composition. In addition, we linked visualization (MALDI MSI) and identification (based on liquid chromatography higher resolution mass spectrometry) of the molecules of interest for the correct biological interpretation of the observed molecular differences within the area in which these molecules are detected. This is of major importance to fully understand the underlying molecular profile of the NSCLC tumor microenvironment.
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Urinary extracellular vesicles (EVs) are an attractive source of biomarkers for urological diseases. A crucial step in biomarker discovery studies is the determination of the variation parameters to perform a sample size calculation. In this way, a biomarker discovery study with sufficient statistical power can be performed to obtain biologically significant biomarkers. Here, a variation study was performed on both the protein and lipid content of urinary EVs of healthy individuals, aged between 52 and 69 years. Ultrafiltration (UF) in combination with size exclusion chromatography (SEC) was used to isolate the EVs from urine. Different experimental variation set-ups were used in this variation study. The calculated standard deviations (SDs) of the 90% least variable peptides and lipids did not exceed 2 and 1.2, respectively. These parameters can be used in a sample size calculation for a well-designed biomarker discovery study at the cargo of EVs.
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In Mycobacterium tuberculosis (Mtb) the detection of single nucleotide polymorphisms (SNPs) is of high importance both for diagnostics, since drug resistance is primarily caused by the acquisition of SNPs in multiple drug targets, and for epidemiological studies in which strain typing is performed by SNP identification. To provide the necessary coverage of clinically relevant resistance profiles and strain types, nucleic acid-based measurement techniques must be able to detect a large number of potential SNPs. Since the Mtb problem is pressing in many resource-poor countries, requiring low-cost point-of-care biosensors, this is a non-trivial technological challenge. This paper presents a proof-of-concept in which we chose simple DNA-DNA hybridization as a sensing principle since this can be transferred to existing low-cost hardware platforms, and we pushed the multiplex boundaries of it. With a custom designed probe set and a physicochemical-driven data analysis it was possible to simultaneously detect the presence of SNPs associated with first- and second-line drug resistance and Mtb strain typing. We have demonstrated its use for the identification of drug resistance and strain type from a panel of phylogenetically diverse clinical strains. Furthermore, reliable detection of the presence of a minority population (<5%) of drug-resistant Mtb was possible.
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DNA Bacteriano/metabolismo , Mycobacterium tuberculosis/genética , Tuberculose/patologia , Antituberculosos/farmacologia , DNA Bacteriano/genética , Humanos , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase Multiplex , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Hibridização de Ácido Nucleico , Polimorfismo de Nucleotídeo Único , Tuberculose/microbiologiaRESUMO
Extracellular vesicles (EVs) have a great potential in clinical applications. However, their isolation from different bodily fluids and their characterisation are currently not optimal or standardised. Here, we report the results of examining the performance of ultrafiltration combined with size exclusion chromatography (UF-SEC) to isolate EVs from urine. The results reveal that UF-SEC is an efficient method and provides high purity. Furthermore, we introduce asymmetrical-flow field-flow fractionation coupled with a UV detector and multi-angle light-scattering detector (AF4/UV-MALS) as a characterisation method and compare it with current methods. We demonstrate that AF4/UV-MALS is a straightforward and reproducible method for determining size, amount and purity of isolated urinary EVs.
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The knowledge of genomic DNA variations in patient samples has a high and increasing value for human diagnostics in its broadest sense. Although many methods and sensors to detect or quantify these variations are available or under development, the number of underlying physico-chemical detection principles is limited. One of these principles is the hybridization of sample target DNA versus nucleic acid probes. We introduce a novel thermodynamics approach and develop a framework to exploit the specific detection capabilities of nucleic acid hybridization, using generic principles applicable to any platform. As a case study, we detect point mutations in the KRAS oncogene on a microarray platform. For the given platform and hybridization conditions, we demonstrate the multiplex detection capability of hybridization and assess the detection limit using thermodynamic considerations; DNA containing point mutations in a background of wild type sequences can be identified down to at least 1% relative concentration. In order to show the clinical relevance, the detection capabilities are confirmed on challenging formalin-fixed paraffin-embedded clinical tumor samples. This enzyme-free detection framework contains the accuracy and efficiency to screen for hundreds of mutations in a single run with many potential applications in molecular diagnostics and the field of personalised medicine.
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DNA/genética , Hibridização de Ácido Nucleico/genética , Mutação Puntual/genética , Genes ras/genética , Humanos , Inclusão em Parafina/métodos , Patologia Molecular , Termodinâmica , Proteínas ras/genéticaRESUMO
In quantitative shotgun proteomic analyses by liquid chromatography and mass spectrometry, a rigid study design is necessary in order to obtain statistically relevant results. Hypothesis testing, sample size calculation and power estimation are fundamental concepts that require consideration upon designing an experiment. For this reason, the reproducibility and variability of the proteomic platform needs to be assessed. In this study, we evaluate the technical (sample preparation), labeling (isobaric labels), and total (biological + technical + labeling + experimental) variability and reproducibility of a workflow that employs a shotgun LC-MS/MS approach in combination with TMT peptide labeling for the quantification of peripheral blood mononuclear cell (PBMC) proteome. We illustrate that the variability induced by TMT labeling is small when compared to the technical variation. The latter is also responsible for a substantial part of the total variation. Prior knowledge about the experimental variability allows for a correct design, a prerequisite for the detection of biologically significant disease-specific differential proteins in clinical proteomics experiments.
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Proteômica/métodos , Proteômica/normas , Espectrometria de Massas em Tandem , Análise por Conglomerados , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Peptídeos/metabolismo , Projetos Piloto , ProteomaRESUMO
The paper describes the inhalation nickel (Ni) exposure of humans via the environment for the regional scale in the EU, together with a tiered approach for assessing additional local exposure from industrial emissions. The approach was designed, in the context of REACH, for the purpose of assessing and controlling emissions and air quality in the neighbourhood of Ni producers and downstream users. Two Derived No Effect Level (DNEL) values for chronic inhalation exposure to total Ni in PM10 (20 and 60ngNi/m(3)) were considered. The value of 20ngNi/m(3) is the current EU air quality guidance value. The value of 60ngNi/m(3) is derived here based on recently published Ni data (Oller et al., 2014). Both values are protective for respiratory toxicity and carcinogenicity but differ in the application of toxicokinetic adjustments and cancer threshold considerations. Estimates of air Ni concentrations at the European regional scale were derived from the database of the European Environment Agency. The 50th and 90th percentile regional exposures were below both DNEL values. To assess REACH compliance at the local scale, measured ambient air data are preferred but are often unavailable. A tiered approach for the use of modelled ambient air concentrations was developed, starting with the application of the default EUSES model and progressing to more sophisticated models. As an example, the tiered approach was applied to 33 EU Ni sulphate producers' and downstream users' sites. Applying the EUSES model demonstrates compliance with a DNEL of 60ngNi/m(3) for the majority of sites, while the value of the refined modelling is demonstrated when a DNEL of 20ngNi/m(3) is considered. The proposed approach, applicable to metals in general, can be used in the context of REACH, for refining the risk characterisation and guiding the selection of risk management measures.
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Poluentes Atmosféricos/análise , Monitoramento Ambiental , Exposição por Inalação/estatística & dados numéricos , Níquel/análise , Europa (Continente) , Humanos , Modelos TeóricosRESUMO
BACKGROUND: For Europe as a whole, data on internal exposure to environmental chemicals do not yet exist. Characterization of the internal individual chemical environment is expected to enhance understanding of the environmental threats to health. OBJECTIVES: We developed and applied a harmonized protocol to collect comparable human biomonitoring data all over Europe. METHODS: In 17 European countries, we measured mercury in hair and cotinine, phthalate metabolites, and cadmium in urine of 1,844 children (5-11 years of age) and their mothers. Specimens were collected over a 5-month period in 2011-2012. We obtained information on personal characteristics, environment, and lifestyle. We used the resulting database to compare concentrations of exposure biomarkers within Europe, to identify determinants of exposure, and to compare exposure biomarkers with health-based guidelines. RESULTS: Biomarker concentrations showed a wide variability in the European population. However, levels in children and mothers were highly correlated. Most biomarker concentrations were below the health-based guidance values. CONCLUSIONS: We have taken the first steps to assess personal chemical exposures in Europe as a whole. Key success factors were the harmonized protocol development, intensive training and capacity building for field work, chemical analysis and communication, as well as stringent quality control programs for chemical and data analysis. Our project demonstrates the feasibility of a Europe-wide human biomonitoring framework to support the decision-making process of environmental measures to protect public health.
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Cotinina/urina , Exposição Ambiental/estatística & dados numéricos , Monitoramento Ambiental/métodos , Poluentes Ambientais/metabolismo , Adulto , Biomarcadores/urina , Cádmio/urina , Criança , Pré-Escolar , Exposição Ambiental/análise , Poluentes Ambientais/análise , Poluentes Ambientais/urina , Europa (Continente)/epidemiologia , Feminino , Cabelo/química , Humanos , Masculino , Mercúrio/análise , Mães , Ácidos Ftálicos/urinaRESUMO
Minor bicycle accidents are defined as "bicycle accidents not involving death or heavily injured persons, implying that possible hospital visits last less than 24 hours". Statistics about these accidents and related injuries are very poor, because they are mostly not reported to police, hospitals or insurance companies. Yet, they form a major share of all bicycle accidents. Official registrations underestimate the number of minor accidents and do not provide cost data, nor the distance cycled. Therefore related policies are hampered by a lack of accurate data. This paper provides more insight into the importance of minor bicycle accidents and reports the frequency, risk and resulting costs of minor bicycle accidents. Direct costs, including the damage to bike and clothes as well as medical costs and indirect costs such as productivity loss and leisure time lost are calculated. We also estimate intangible costs of pain and psychological suffering and costs for other parties involved in the accident. Data were collected during the SHAPES project using several electronic surveys. The weekly prospective registration that lasted a year, covered 1187 persons that cycled 1,474,978 km. 219 minor bicycle accidents were reported. Resulting in a frequency of 148 minor bicycle accidents per million kilometres. We analyzed the economic costs related to 118 minor bicycle accidents in detail. The average total cost of these accidents is estimated at 841 euro (95% CI: 579-1205) per accident or 0.125 euro per kilometre cycled. Overall, productivity loss is the most important component accounting for 48% of the total cost. Intangible costs, which in past research were mostly neglected, are an important burden related to minor bicycle accidents (27% of the total cost). Even among minor accidents there are important differences in the total cost depending on the severity of the injury.