RESUMEN
Testing of large populations for virus infection is now a reality worldwide due to the coronavirus (SARS-CoV-2) pandemic. The demand for SARS-CoV-2 testing using alternatives other than PCR led to the development of mass spectrometry (MS)-based assays. However, MS for SARS-CoV-2 large-scale testing have some downsides, including complex sample preparation and slow data analysis. Here, we describe a high-throughput targeted proteomics method to detect SARS-CoV-2 directly from nasopharyngeal and oropharyngeal swabs. This strategy employs fully automated sample preparation mediated by magnetic particles, followed by detection of SARS-CoV-2 nucleoprotein peptides by turbulent flow chromatography coupled with tandem mass spectrometry.
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COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Prueba de COVID-19 , Humanos , Pandemias , Espectrometría de Masas en Tándem/métodosRESUMEN
Signal variation is a common drawback in untargeted metabolomics using liquid chromatography-mass spectrometry (LC-MS), mainly due to the complexity of biological matrices and reduced sample preparation, which results in the accumulation of sample components in the column and the ion source. Here we propose a simple, easy to implement approach to improve data quality in untargeted metabolomics by LC-MS. This approach involves the use of a divert valve to direct the column effluent to waste at the beginning of the chromatographic run and during column cleanup and equilibration, in combination with longer column cleanups in between injections. Our approach was tested using urine samples collected from patients after renal transplantation. Analytical responses were contrasted before and after introducing these modifications by analyzing a batch of untargeted metabolomics data. A significant improvement in peak area repeatability was observed for the quality controls, with relative standard deviations (RSDs) for several metabolites decreasing from â¼60% to â¼10% when our approach was introduced. Similarly, RSDs of peak areas for internal standards improved from â¼40% to â¼10%. Furthermore, calibrant solutions were more consistent after introducing these modifications when comparing peak areas of solutions injected at the beginning and the end of each analytical sequence. Therefore, we recommend the use of a divert valve and extended column cleanup as a powerful strategy to improve data quality in untargeted metabolomics, especially for very complex types of samples where minimum sample preparation is required, such as in this untargeted metabolomics study with urine from renal transplanted patients.
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Cromatografía Liquida , Exactitud de los Datos , Espectrometría de Masas , Metabolómica , Urinálisis , Humanos , Urinálisis/métodos , Urinálisis/normas , Orina/químicaRESUMEN
The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is pressing public health systems around the world, and large population testing is a key step to control this pandemic disease. Here, we develop a high-throughput targeted proteomics assay to detect SARS-CoV-2 nucleoprotein peptides directly from nasopharyngeal and oropharyngeal swabs. A modified magnetic particle-based proteomics approach implemented on a robotic liquid handler enables fully automated preparation of 96 samples within 4 hours. A TFC-MS system allows multiplexed analysis of 4 samples within 10 min, enabling the processing of more than 500 samples per day. We validate this method qualitatively (Tier 3) and quantitatively (Tier 1) using 985 specimens previously analyzed by real-time RT-PCR, and detect up to 84% of the positive cases with up to 97% specificity. The presented strategy has high sample stability and should be considered as an option for SARS-CoV-2 testing in large populations.
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Prueba de COVID-19/métodos , Técnicas de Laboratorio Clínico , Espectrometría de Masas/métodos , Humanos , Nasofaringe/virología , Orofaringe/virología , Proteómica , SARS-CoV-2/aislamiento & purificación , Sensibilidad y Especificidad , Proteínas ViralesRESUMEN
Atherosclerotic plaque development is closely associated with the hemodynamic forces applied to endothelial cells (ECs). Among these, shear stress (SS) plays a key role in disease development since changes in flow intensity and direction could stimulate an atheroprone or atheroprotective phenotype. ECs under low or oscillatory SS (LSS) show upregulation of inflammatory, adhesion, and cellular permeability molecules. On the contrary, cells under high or laminar SS (HSS) increase their expression of protective and anti-inflammatory factors. The mechanism behind SS regulation of an atheroprotective phenotype is not completely elucidated. Here we used proteomics and metabolomics to better understand the changes in endothelial cells (human umbilical vein endothelial cells) under in vitro LSS and HSS that promote an atheroprone or atheroprotective profile and how these modifications can be connected to atherosclerosis development. Our data showed that lipid metabolism, in special cholesterol metabolism, was downregulated in cells under LSS. The low-density lipoprotein receptor (LDLR) showed significant alterations both at the quantitative expression level as well as regarding posttranslational modifications. Under LSS, LDLR was seen at lower concentrations and with a different glycosylation profile. Finally, modulating LDLR with atorvastatin led to the recapitulation of a HSS metabolic phenotype in EC under LSS. Altogether, our data suggest that there is significant modulation of lipid metabolism in endothelial cells under different SS intensities and that this could contribute to the atheroprone phenotype of LSS. Statin treatment was able to partially recover the protective profile of these cells.
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Aterosclerosis/metabolismo , Hemodinámica , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Metabolismo de los Lípidos , Lipidómica/métodos , Mecanotransducción Celular , Proteómica/métodos , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Atorvastatina/farmacología , Células Cultivadas , Colesterol/metabolismo , Glicosilación , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Mecanotransducción Celular/efectos de los fármacos , Fenotipo , Placa Aterosclerótica , Procesamiento Proteico-Postraduccional , Receptores de LDL/metabolismo , Flujo Sanguíneo Regional , Estrés MecánicoRESUMEN
It has been recently proposed that exposure to polychlorinated biphenyls (PCBs) is a risk factor to type 2 diabetes mellitus (DM2). We investigated this hypothesis using long-term in vivo PCB126 exposure to rats addressing metabolic, cellular and proteomic parameters. Male Wistar rats were exposed to PCB126 (0.1, 1 or 10 µg/kg of body weight/day; for 15 days) or vehicle by intranasal instillation. Systemic alterations were quantified by body weight, insulin and glucose tolerance, and blood biochemical profile. Pancreatic toxicity was measured by inflammatory parameters, cell viability and cycle, free radical generation, and proteomic profile on islets of Langerhans. In vivo PCB126 exposure enhanced the body weight gain, impaired insulin sensitivity, reduced adipose tissue deposit, and elevated serum triglycerides, cholesterol, and insulin levels. Inflammatory parameters in the pancreas and cell morphology, viability and cycle were not altered in islets of Langerhans. Nevertheless, in vivo PCB126 exposure increased free radical generation and modified the expression of proteins related to oxidative stress on islets of Langerhans, which are indicative of early ß-cell failure. Data herein obtained show that long-term in vivo PCB126 exposure through intranasal route induced alterations on islets of Langerhans related to early end points of DM2.
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Islotes Pancreáticos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Bifenilos Policlorados/toxicidad , Proteoma/efectos de los fármacos , Tejido Adiposo/metabolismo , Administración Intranasal , Animales , Peso Corporal/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colesterol/sangre , Glucosa/metabolismo , Insulina/sangre , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Óxido Nítrico/metabolismo , Ratas , Ratas Wistar , Triglicéridos/sangre , Regulación hacia Arriba/efectos de los fármacosRESUMEN
OBJECTIVE: To analyze the seminal plasma proteome and biological functions associated with sperm functional alterations. DESIGN: Cross-sectional study. SETTING: University andrology and research laboratories. PATIENT(S): A total of 156 normozoospermic men. INTERVENTION(S): Sperm mitochondrial activity, acrosome integrity, and DNA fragmentation were evaluated in a semen aliquot. Remaining semen was centrifuged, and seminal plasma was utilized for proteomic analysis (liquid chromatography-tandem mass spectrometry). Patients were divided into percentiles (15%) to form the following groups: substudy 1, high (control, n = 26) and low (study, n = 23) sperm mitochondrial activity; substudy 2, high (control, n = 23) and low (study, n = 22) sperm acrosome integrity; and substudy 3, low (control, n = 22) and high (study, n = 22) sperm DNA fragmentation. Groups were compared using univariate and multivariate analyses. Differentially expressed proteins were used for functional enrichment analysis. MAIN OUTCOME MEASURE(S): Seminal plasma proteome and postgenomic pathways are associated with several sperm functional traits. RESULT(S): In total, 506, 493, and 464 proteins were observed in substudies 1, 2, and 3, respectively. Enriched functions in substudy 1 were intramolecular oxidoreductase activity, aminoglycans catabolism, endopeptidases inhibition, lysosomes, and acute-phase response (study group). In substudy 2, main enriched functions were phospholipase inhibition, arachidonic acid metabolism, exocytosis, regulation of acute inflammation, response to hydrogen peroxide, and lysosomal transport (study group). In substudy 3, enriched functions were prostaglandin biosynthesis and fatty acid binding (study group). We proposed eight, six, and eight seminal biomarkers for substudies 1, 2, and 3, respectively. CONCLUSION(S): Seminal plasma proteome reflects sperm mitochondrial activity reduction, acrosome damage, and DNA fragmentation, with several postgenomic functions related to these alterations.
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Semen/metabolismo , Proteínas de Plasma Seminal/metabolismo , Espermatozoides/metabolismo , Acrosoma/metabolismo , Acrosoma/patología , Adulto , Biomarcadores/metabolismo , Cromatografía Liquida , Estudios Transversales , Daño del ADN , ADN Mitocondrial/metabolismo , Análisis Discriminante , Fertilidad , Humanos , Análisis de los Mínimos Cuadrados , Modelos Lineales , Modelos Logísticos , Masculino , Persona de Mediana Edad , Mitocondrias/metabolismo , Mitocondrias/patología , Análisis Multivariante , Proteómica/métodos , Espermatozoides/patología , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Chronic hepatitis B (CHB) virus infection is a major cause of hepatocellular carcinoma (HCC), as late diagnosis is the main factor for the poor survival of patients. There is an urgent need for accurate biomarkers for early diagnosis of HCC. The aim of the study was to explore the serum lipidome profiles of hepatitis B-related HCC to identify potential diagnostic biomarkers. METHODS: An ultraperformance liquid chromatography mass spectrometry (UPLC-MS) lipidomic method was used to characterize serum profiles from HCC (n = 32), liver cirrhosis (LC) (n = 30), CHB (n = 25), and healthy subjects (n = 34). Patients were diagnosed by clinical laboratory and imaging evidence and all presented with CHB while healthy controls had normal liver function and no infectious diseases. RESULTS: The UPLC-MS-based serum lipidomic profile provided more accurate diagnosis for LC patients than conventional alpha-fetoprotein (AFP) detection. HCC patients were discriminated from LC with 78 % sensitivity and 64 % specificity. In comparison, AFP showed sensitivity and specificity of 38 % and 93 %, respectively. HCC was differentiated from CHB with 100 % sensitivity and specificity using the UPLC-MS approach. Identified lipids comprised glycerophosphocolines, glycerophosphoserines and glycerophosphoinositols. CONCLUSIONS: UPLC-MS lipid profiling proved to be an efficient and convenient tool for diagnosis and screening of HCC in a high-risk population.
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Biomarcadores de Tumor/sangre , Carcinoma Hepatocelular/virología , Cromatografía de Gases y Espectrometría de Masas/métodos , Hepatitis B Crónica/diagnóstico , Lípidos/sangre , Neoplasias Hepáticas/virología , Adulto , Anciano , Carcinoma Hepatocelular/sangre , Carcinoma Hepatocelular/diagnóstico , Diagnóstico Diferencial , Detección Precoz del Cáncer , Femenino , Hepatitis B Crónica/sangre , Humanos , Cirrosis Hepática/sangre , Cirrosis Hepática/diagnóstico , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/diagnóstico , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
INTRODUCTION: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that affects around 1% of the human population worldwide. RA diagnosis can be difficult as there is no definitive test for its detection. Therefore, the aim of this study was to identify biomarkers that could be used for RA diagnosis. METHODS: Sera from a collagen-induced arthritis mouse model were used to select potential biomarkers for RA diagnosis by phage display technology. In silico and in vitro analyses were performed to characterize and validate the selected peptides. Samples were classified into three groups: RA; two other immune-mediated rheumatic diseases (systemic lupus erythematosus (SLE) and ankylosing spondylitis (AS)); and healthy controls (HC). Enzyme-linked immunosorbent assay (ELISA) was carried out to determine antibody levels, and diagnostic parameters were determined by constructing receiver operating characteristic curves. Mass spectrometry and Western blot were performed to identify the putative autoantigen that was mimicked by a highly reactive mimotope. RESULTS: After three rounds of selection, 14 clones were obtained and tested for immunoreactivity analysis against sera from RA and HC groups. The phage-fused peptide with the highest immunoreactivity (M12) was synthesized, and was able to efficiently discriminate RA patients from SLE, AS and HCs (p < 0.0001) by ELISA. The specificity and sensitivity of anti-M12 antibodies for RA diagnosis were 91 % and 84.3 %, respectively. The M12 peptide was identified as one that mimics a predicted antigenic site of the carbonic anhydrase III (CAIII) protein, a ubiquitous biomarker that has been identified in patients with other diseases. CONCLUSION: M12 is the first peptide associated with the CAIII protein that may be used as an antigen for antibody detection to aid in RA diagnosis with high sensitivity and specificity.
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Artritis Reumatoide/sangre , Artritis Reumatoide/diagnóstico , Anhidrasa Carbónica III/sangre , Técnicas de Visualización de Superficie Celular/métodos , Modelos Animales de Enfermedad , Imitación Molecular/fisiología , Adulto , Anciano , Secuencia de Aminoácidos , Animales , Artritis Reumatoide/genética , Anhidrasa Carbónica III/genética , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos DBA , Persona de Mediana Edad , Datos de Secuencia Molecular , Estructura Secundaria de ProteínaRESUMEN
OBJECTIVE: To study the seminal plasma proteome in association with semen lipid peroxidation levels in men with normal semen parameters. DESIGN: Cross-sectional study. SETTING: University andrology and research laboratories. PATIENT(S): A total of 156 normozoospermic men. INTERVENTION(S): Seminal lipid peroxidation levels were assessed in individual samples through thiobarbituric acid reactive substances quantification. Subsequently, lipid peroxidation data were used to divide the samples into the experimental groups: low lipid peroxidation levels (control group, bottom 15%, n = 23) and high lipid peroxidation levels (study group, top 15%, n = 23). Seminal plasma proteins from these groups were pooled (four pools per group, with biological variation between the pools) and used for a shotgun proteomic analysis using a liquid chromatography-tandem mass spectrometry approach. Quantitative data were used for univariate (unpaired Student's t test) and multivariate (partial least-squares discriminant analysis, logistic regression, and discriminant analyses) statistical analyses. Significant proteins were also used for functional enrichment analysis. MAIN OUTCOME MEASURE(S): Seminal plasma protein profile and postgenomic pathways of seminal plasma are associated with seminal lipid peroxidation levels. RESULT(S): In total, 629 proteins were quantified in seminal plasma. Of these, 23 proteins were absent or underexpressed and 71 were exclusive or overexpressed in the study group. The main enriched functions in association with seminal lipid peroxidation were unsaturated fatty acids biosynthesis, oxidants and antioxidants activity, cellular response to heat stress, and immune response. Moreover, we suggested mucin-5B as a potential biomarker of semen oxidative stress. CONCLUSION(S): The seminal plasma proteome does reflect semen lipid peroxidation status and, thus, oxidative stress.
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Estrés Oxidativo/fisiología , Proteoma/biosíntesis , Semen/metabolismo , Proteínas de Plasma Seminal/biosíntesis , Adulto , Estudios Transversales , Humanos , Peroxidación de Lípido/fisiología , Masculino , Persona de Mediana Edad , Proteoma/genética , Análisis de Semen/métodos , Proteínas de Plasma Seminal/genética , Adulto JovenRESUMEN
We present here the effect of heavy metals and of different light intensities on the biosynthesis of fatty acids and pigments in the macroalga Gracilaria tenuistipitata (var. liui Zhang & Xia). In order to verify the fatty acid content, gas chromatography with flame ionization detection (GC-FID) was employed. Pigments (major carotenoids and chlorophyl-a) were monitored by liquid chromatography with diode array detection (HPLC-DAD). Cultures of G. tenuistipitata were exposed to cadmium (Cd2+, 200 ppb) and copper (Cu2+, 200 ppb), as well as to different light conditions (low light: 100 µmol.photons.m-2.s-1, or high light: 1000 µmol.photons.m-2.s-1). Cd2+ and Cu2+ increased the saturated and monounsaturated fatty acid content [14:0, 16:0, 18:0, 18:1 (n-7) and 18:1 (n-9)] and all major pigments (violaxanthin, antheraxanthin, lutein, zeaxanthin, chlorophyll-a and β-carotene). Both heavy metals decreased the levels of polyunsaturated fatty acids (PUFA) [18:2 (n-6), 18:3 (n-6), 18:5 (n-4), 20:4 (n-6), 20:5 (n-3), 22:6 (n-3)]. G. tenuistipitata cultures were exposed to high light intensity for five days and no statistically significant differences were observed in the content of fatty acids. On the other hand, the levels of pigments rose markedly for chlorophyll-a and all of the carotenoids studied.
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Drastic environmental conditions such as elevated temperature, abrupt pH variation, low turbulence, and high nutrient inputs can enhance the development of toxic cyanobacterial blooms in lakes and reservoirs. This study describes the occurrence of four microcystin variants (MC) in a bloom in the eutrophic reservoir Billings, in São Paulo City. The bloom sample was collected in October 2003, and Microcystis were the main genus found. The MC were separated and purified by reverse phase high performance liquid chromatography (RP-HPLC). Their structures were elucidated by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and four MC variants were determined: MC-RR, MC-LR, MC-YR, and MC-hRhR. MC-hRhR is described for the first time as a new variant of MC with two homoarginines at positions 2 and 4 in its structure. ESI-MS/MS analysis thus provides a powerful and convenient tool for the determination of variants of MC. These results represent an important contribution to the knowledge of the biochemistry of toxic cyanobacteria and their toxins, specifically in São Paulo State.
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Cianobacterias/aislamiento & purificación , Cianobacterias/metabolismo , Monitoreo del Ambiente/métodos , Eutrofización/fisiología , Péptidos Cíclicos/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Microbiología del Agua , Cianobacterias/clasificación , Microcistinas , Contaminantes del Agua/análisisRESUMEN
The presence of microcystins (MCY) in the cyanobacteria Microcystis panniformis Komárek et al. is reported for the first time. This strain of cyanobacterium has been isolated from Barra Bonita, an eutrophicated water reservoir in São Paulo state, Brazil. The identification of M. panniformis was confirmed by both traditional morphological analysis and the phycocyanin intergenic spacer sequences. MCY-LR and [Asp(3)]-MCY-LR were identified in this strain after HPLC purification and extensive ESI-MS/MS analysis. Their levels in this strain were determined by HPLC and ranged from 0.25 to 2.75 and 0.08 to 0.75 fmol/cell, respectively. Analyzing the levels of MCY-LR and [Asp(3)]-MCY-LR in different times during the light:dark (L:D) cycle, it was found that levels of MCYs per cell were at least threefold as high during the day-phase than during the night-phase. This may be associated to the biological clock since prokaryotic cyanobacteria express robust circadian (daily) rhythms under the control of a timing mechanism that is independent of the cell division cycle. Our findings also showed the same pattern under light:light (L:L) cycle.