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1.
Nat Commun ; 11(1): 3903, 2020 08 06.
Artículo en Inglés | MEDLINE | ID: mdl-32764543

RESUMEN

Top-down mass spectrometry (MS)-based proteomics provides a comprehensive analysis of proteoforms to achieve a proteome-wide understanding of protein functions. However, the MS detection of low-abundance proteins from blood remains an unsolved challenge due to the extraordinary dynamic range of the blood proteome. Here, we develop an integrated nanoproteomics method coupling peptide-functionalized superparamagnetic nanoparticles (NPs) with top-down MS for the enrichment and comprehensive analysis of cardiac troponin I (cTnI), a gold-standard cardiac biomarker, directly from serum. These NPs enable the sensitive enrichment of cTnI (<1 ng/mL) with high specificity and reproducibility, while simultaneously depleting highly abundant proteins such as human serum albumin (>1010 more abundant than cTnI). We demonstrate that top-down nanoproteomics can provide high-resolution proteoform-resolved molecular fingerprints of diverse cTnI proteoforms to establish proteoform-pathophysiology relationships. This scalable and reproducible antibody-free strategy can generally enable the proteoform-resolved analysis of low-abundance proteins directly from serum to reveal previously unachievable molecular details.


Asunto(s)
Análisis Químico de la Sangre/métodos , Proteínas Sanguíneas/análisis , Espectrometría de Masas/métodos , Proteómica/métodos , Troponina I/sangre , Biomarcadores/sangre , Humanos , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/ultraestructura , Nanotecnología , Procesamiento Proteico-Postraduccional , Proteoma/análisis , Reproducibilidad de los Resultados , Albúmina Sérica Humana/análisis
2.
Nat Commun ; 11(1): 3850, 2020 07 31.
Artículo en Inglés | MEDLINE | ID: mdl-32737322

RESUMEN

Resolving the distribution of specific proteins at the nanoscale in the ultrastructural context of the cell is a major challenge in fluorescence microscopy. We report the discovery of a new principle for an optical contrast equivalent to electron microscopy (EM) which reveals the ultrastructural context of the cells with a conventional confocal microscope. By decrowding the intracellular space through 13 to 21-fold physical expansion while simultaneously retaining the proteins, bulk (pan) labeling of the proteome resolves local protein densities and reveals the cellular nanoarchitecture by standard light microscopy.


Asunto(s)
Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , Proteoma/análisis , Coloración y Etiquetado/métodos , Acrilamidas/química , Reactivos de Enlaces Cruzados/química , Colorantes Fluorescentes/química , Células HeLa , Humanos , Hidrogeles/química , Espacio Intracelular/química , Succinimidas/química , Adhesión del Tejido/métodos
3.
Nat Commun ; 11(1): 3793, 2020 07 30.
Artículo en Inglés | MEDLINE | ID: mdl-32732981

RESUMEN

Reproducible research is the bedrock of experimental science. To enable the deployment of large-scale proteomics, we assess the reproducibility of mass spectrometry (MS) over time and across instruments and develop computational methods for improving quantitative accuracy. We perform 1560 data independent acquisition (DIA)-MS runs of eight samples containing known proportions of ovarian and prostate cancer tissue and yeast, or control HEK293T cells. Replicates are run on six mass spectrometers operating continuously with varying maintenance schedules over four months, interspersed with ~5000 other runs. We utilise negative controls and replicates to remove unwanted variation and enhance biological signal, outperforming existing methods. We also design a method for reducing missing values. Integrating these computational modules into a pipeline (ProNorM), we mitigate variation among instruments over time and accurately predict tissue proportions. We demonstrate how to improve the quantitative analysis of large-scale DIA-MS data, providing a pathway toward clinical proteomics.


Asunto(s)
Espectrometría de Masas/métodos , Proteoma/análisis , Proteómica/métodos , Biomarcadores de Tumor/análisis , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Masculino , Neoplasias Ováricas , Neoplasias de la Próstata , Reproducibilidad de los Resultados , Saccharomyces cerevisiae
4.
Nat Commun ; 11(1): 4332, 2020 08 28.
Artículo en Inglés | MEDLINE | ID: mdl-32859902

RESUMEN

The group of enteroviruses contains many important pathogens for humans, including poliovirus, coxsackievirus, rhinovirus, as well as newly emerging global health threats such as EV-A71 and EV-D68. Here, we describe an unbiased, system-wide and time-resolved analysis of the proteome and phosphoproteome of human cells infected with coxsackievirus B3. Of the ~3,200 proteins quantified throughout the time course, a large amount (~25%) shows a significant change, with the majority being downregulated. We find ~85% of the detected phosphosites to be significantly regulated, implying that most changes occur at the post-translational level. Kinase-motif analysis reveals temporal activation patterns of certain protein kinases, with several CDKs/MAPKs immediately active upon the infection, and basophilic kinases, ATM, and ATR engaging later. Through bioinformatics analysis and dedicated experiments, we identify mTORC1 signalling as a major regulation network during enterovirus infection. We demonstrate that inhibition of mTORC1 activates TFEB, which increases expression of lysosomal and autophagosomal genes, and that TFEB activation facilitates the release of virions in extracellular vesicles via secretory autophagy. Our study provides a rich framework for a system-level understanding of enterovirus-induced perturbations at the protein and signalling pathway levels, forming a base for the development of pharmacological inhibitors to treat enterovirus infections.


Asunto(s)
Infecciones por Coxsackievirus/metabolismo , Interacciones Huésped-Patógeno/fisiología , Proteoma/análisis , Animales , Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Línea Celular , Supervivencia Celular , Enterovirus/fisiología , Enterovirus Humano B/fisiología , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Fosforilación , Transducción de Señal , Proteínas Virales/metabolismo
5.
PLoS One ; 15(7): e0235904, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32663208

RESUMEN

Pancreatic ductal adenocarcinoma is one of the most aggressive types of cancer. Certain proteins in the tumor microenvironment have attracted considerable attention owing to their association with tumor invasion and metastasis. Here, we used proteomics to identify proteins associated with lymph-node metastasis, which is one of the prognostic factors. We selected lymph node metastasis-positive and -negative patients (n = 5 each) who underwent pancreatectomy between 2005 and 2015 and subjected to comprehensive proteomic profiling of tumor stroma. A total of 490 proteins were detected by mass spectrometry. Software analysis revealed that nine of these proteins were differentially expressed between the two patient groups. We focused on hemopexin and ferritin light chain based on immunohistochemistry results. We assessed the clinicopathological data of 163 patients and found that hemopexin expression was associated with UICC N2 (p = 0.0399), lymph node ratio (p = 0.0252), venous invasion (p = 0.0096), and lymphatic invasion (p = 0.0232). Notably, in vitro assays showed that hemopexin promotes invasion of the pancreatic cancer cells. Our findings suggest that hemopexin is a lymph node metastasis-associated protein that could potentially serve as a useful therapeutic target or biomarker of pancreatic ductal adenocarcinoma.


Asunto(s)
Carcinoma Ductal Pancreático/patología , Hemopexina/metabolismo , Neoplasias Pancreáticas/patología , Anciano , Apoferritinas/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Movimiento Celular , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Inmunohistoquímica , Metástasis Linfática , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Neoplasias Pancreáticas/metabolismo , Pronóstico , Proteoma/análisis , Proteómica/métodos , Espectrometría de Masas en Tándem
6.
Proc Natl Acad Sci U S A ; 117(29): 17094-17103, 2020 07 21.
Artículo en Inglés | MEDLINE | ID: mdl-32611817

RESUMEN

Declining ejaculate performance with male age is taxonomically widespread and has broad fitness consequences. Ejaculate success requires fully functional germline (sperm) and soma (seminal fluid) components. However, some aging theories predict that resources should be preferentially diverted to the germline at the expense of the soma, suggesting differential impacts of aging on sperm and seminal fluid and trade-offs between them or, more broadly, between reproduction and lifespan. While harmful effects of male age on sperm are well known, we do not know how much seminal fluid deteriorates in comparison. Moreover, given the predicted trade-offs, it remains unclear whether systemic lifespan-extending interventions could ameliorate the declining performance of the ejaculate as a whole. Here, we address these problems using Drosophila melanogaster. We demonstrate that seminal fluid deterioration contributes to male reproductive decline via mating-dependent mechanisms that include posttranslational modifications to seminal proteins and altered seminal proteome composition and transfer. Additionally, we find that sperm production declines chronologically with age, invariant to mating activity such that older multiply mated males become infertile principally via reduced sperm transfer and viability. Our data, therefore, support the idea that both germline and soma components of the ejaculate contribute to male reproductive aging but reveal a mismatch in their aging patterns. Our data do not generally support the idea that the germline is prioritized over soma, at least, within the ejaculate. Moreover, we find that lifespan-extending systemic down-regulation of insulin signaling results in improved late-life ejaculate performance, indicating simultaneous amelioration of both somatic and reproductive aging.


Asunto(s)
Envejecimiento , Drosophila melanogaster , Proteínas de Plasma Seminal , Espermatozoides , Envejecimiento/genética , Envejecimiento/fisiología , Animales , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Femenino , Fertilidad/genética , Fertilidad/fisiología , Infertilidad Masculina/genética , Infertilidad Masculina/fisiopatología , Masculino , Proteoma/análisis , Proteoma/genética , Proteoma/fisiología , Proteínas de Plasma Seminal/análisis , Proteínas de Plasma Seminal/fisiología , Conducta Sexual Animal/fisiología , Espermatozoides/química , Espermatozoides/fisiología
7.
PLoS One ; 15(7): e0236148, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32692761

RESUMEN

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is an unexplained chronic, debilitating illness characterized by fatigue, sleep disturbances, cognitive dysfunction, orthostatic intolerance and gastrointestinal problems. Using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), we analyzed the plasma proteomes of 39 ME/CFS patients and 41 healthy controls. Logistic regression models, with both linear and quadratic terms of the protein levels as independent variables, revealed a significant association between ME/CFS and the immunoglobulin heavy variable (IGHV) region 3-23/30. Stratifying the ME/CFS group based on self-reported irritable bowel syndrome (sr-IBS) status revealed a significant quadratic effect of immunoglobulin lambda constant region 7 on its association with ME/CFS with sr-IBS whilst IGHV3-23/30 and immunoglobulin kappa variable region 3-11 were significantly associated with ME/CFS without sr-IBS. In addition, we were able to predict ME/CFS status with a high degree of accuracy (AUC = 0.774-0.838) using a panel of proteins selected by 3 different machine learning algorithms: Lasso, Random Forests, and XGBoost. These algorithms also identified proteomic profiles that predicted the status of ME/CFS patients with sr-IBS (AUC = 0.806-0.846) and ME/CFS without sr-IBS (AUC = 0.754-0.780). Our findings are consistent with a significant association of ME/CFS with immune dysregulation and highlight the potential use of the plasma proteome as a source of biomarkers for disease.


Asunto(s)
Linfocitos B/inmunología , Biomarcadores/sangre , Síndrome de Fatiga Crónica/sangre , Síndrome de Fatiga Crónica/inmunología , Proteoma/análisis , Linfocitos B/patología , Estudios de Casos y Controles , Síndrome de Fatiga Crónica/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Espectrometría de Masas en Tándem
8.
N Engl J Med ; 383(4): 321-333, 2020 07 23.
Artículo en Inglés | MEDLINE | ID: mdl-32706533

RESUMEN

BACKGROUND: Environmental enteric dysfunction (EED) is an enigmatic disorder of the small intestine that is postulated to play a role in childhood undernutrition, a pressing global health problem. Defining the incidence of this disorder, its pathophysiological features, and its contribution to impaired linear and ponderal growth has been hampered by the difficulty in directly sampling the small intestinal mucosa and microbial community (microbiota). METHODS: In this study, among 110 young children (mean age, 18 months) with linear growth stunting who were living in an urban slum in Dhaka, Bangladesh, and had not benefited from a nutritional intervention, we performed endoscopy in 80 children who had biopsy-confirmed EED and available plasma and duodenal samples. We quantified the levels of 4077 plasma proteins and 2619 proteins in duodenal biopsy samples obtained from these children. The levels of bacterial strains in microbiota recovered from duodenal aspirate from each child were determined with the use of culture-independent methods. In addition, we obtained 21 plasma samples and 27 fecal samples from age-matched healthy children living in the same area. Young germ-free mice that had been fed a Bangladeshi diet were colonized with bacterial strains cultured from the duodenal aspirates. RESULTS: Of the bacterial strains that were obtained from the children, the absolute levels of a shared group of 14 taxa (which are not typically classified as enteropathogens) were negatively correlated with linear growth (length-for-age z score, r = -0.49; P = 0.003) and positively correlated with duodenal proteins involved in immunoinflammatory responses. The representation of these 14 duodenal taxa in fecal microbiota was significantly different from that in samples obtained from healthy children (P<0.001 by permutational multivariate analysis of variance). Enteropathy of the small intestine developed in gnotobiotic mice that had been colonized with cultured duodenal strains obtained from children with EED. CONCLUSIONS: These results provide support for a causal relationship between growth stunting and components of the small intestinal microbiota and enteropathy and offer a rationale for developing therapies that target these microbial contributions to EED. (Funded by the Bill and Melinda Gates Foundation and others; ClinicalTrials.gov number, NCT02812615.).


Asunto(s)
Duodeno/microbiología , Microbioma Gastrointestinal , Trastornos del Crecimiento/microbiología , Trastornos de la Nutrición del Lactante/complicaciones , Animales , Bacterias/aislamiento & purificación , Bangladesh , Duodenoscopía , Duodeno/patología , Enfermedades Ambientales/complicaciones , Heces/microbiología , Femenino , Vida Libre de Gérmenes , Crecimiento , Trastornos del Crecimiento/etiología , Humanos , Lactante , Enfermedades Inflamatorias del Intestino/complicaciones , Factor I del Crecimiento Similar a la Insulina/análisis , Enfermedades Intestinales/complicaciones , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis Multivariante , Proteínas Asociadas a Pancreatitis/análisis , Proteoma/análisis
9.
PLoS One ; 15(7): e0230400, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32639965

RESUMEN

Alterations in the cortico-cerebellar-thalamic-cortical circuit might underlie the diversity of symptoms in schizophrenia. However, molecular changes in cerebellar neuronal circuits, part of this network, have not yet been fully determined. Using LC-MS/MS, we screened altered candidates in pooled grey matter of cerebellum from schizophrenia subjects who committed suicide (n = 4) and healthy individuals (n = 4). Further validation by immunoblotting of three selected candidates was performed in two cohorts comprising schizophrenia (n = 20), non-schizophrenia suicide (n = 6) and healthy controls (n = 21). We found 99 significantly altered proteins, 31 of them previously reported in other brain areas by proteomic studies. Transport function was the most enriched category, while cell communication was the most prevalent function. For validation, we selected the vacuolar proton pump subunit 1 (VPP1), from transport, and two EF-hand calcium-binding proteins, calmodulin and parvalbumin, from cell communication. All candidates showed significant changes in schizophrenia (n = 7) compared to controls (n = 7). VPP1 was altered in the non-schizophrenia suicide group and increased levels of parvalbumin were linked to antipsychotics. Further validation in an independent cohort of non-suicidal chronic schizophrenia subjects (n = 13) and non-psychiatric controls (n = 14) showed that parvalbumin was increased, while calmodulin was decreased in schizophrenia. Our findings provide evidence of calcium-binding protein dysregulation in the cerebellum in schizophrenia, suggesting an impact on normal calcium-dependent synaptic functioning of cerebellar circuits. Our study also links VPP1 to suicide behaviours, suggesting a possible impairment in vesicle neurotransmitter refilling and release in these phenotypes.


Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Cerebelo/metabolismo , Esquizofrenia/patología , Adulto , Calmodulina/metabolismo , Estudios de Casos y Controles , Cromatografía Líquida de Alta Presión , Regulación hacia Abajo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Parvalbúminas/metabolismo , Proteoma/análisis , Esquizofrenia/metabolismo , Intento de Suicidio , Espectrometría de Masas en Tándem , Regulación hacia Arriba
10.
Nat Commun ; 11(1): 3581, 2020 07 14.
Artículo en Inglés | MEDLINE | ID: mdl-32665645

RESUMEN

We still know very little about how the human immune system responds to SARS-CoV-2. Here we construct a SARS-CoV-2 proteome microarray containing 18 out of the 28 predicted proteins and apply it to the characterization of the IgG and IgM antibodies responses in the sera from 29 convalescent patients. We find that all these patients had IgG and IgM antibodies that specifically bind SARS-CoV-2 proteins, particularly the N protein and S1 protein. Besides these proteins, significant antibody responses to ORF9b and NSP5 are also identified. We show that the S1 specific IgG signal positively correlates with age and the level of lactate dehydrogenase (LDH) and negatively correlates with lymphocyte percentage. Overall, this study presents a systemic view of the SARS-CoV-2 specific IgG and IgM responses and provides insights to aid the development of effective diagnostic, therapeutic and vaccination strategies.


Asunto(s)
Anticuerpos Antivirales/inmunología , Betacoronavirus/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Análisis por Matrices de Proteínas/métodos , Adulto , Infecciones por Coronavirus/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , L-Lactato Deshidrogenasa/sangre , Masculino , Persona de Mediana Edad , Proteínas de la Nucleocápside/inmunología , Pandemias , Neumonía Viral/inmunología , Proteoma/análisis , Proteoma/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Proteínas no Estructurales Virales/inmunología
11.
PLoS One ; 15(6): e0231681, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32555742

RESUMEN

Eudiplozoon nipponicum (Goto, 1891) is a hematophagous monogenean ectoparasite which inhabits the gills of the common carp (Cyprinus carpio). Heavy infestation can lead to anemia and in conjunction with secondary bacterial infections cause poor health and eventual death of the host. This study is based on an innovative approach to protein localization which has never been used in parasitology before. Using laser capture microdissection, we dissected particular areas of the parasite body without contaminating the samples by surrounding tissue and in combination with analysis by mass spectrometry obtained tissue-specific proteomes of tegument, intestine, and parenchyma of our model organism, E. nipponicum. We successfully verified the presence of certain functional proteins (e.g. cathepsin L) in tissues where their presence was expected (intestine) and confirmed that there were no traces of these proteins in other tissues (tegument and parenchyma). Additionally, we identified a total of 2,059 proteins, including 72 peptidases and 33 peptidase inhibitors. As expected, the greatest variety was found in the intestine and the lowest variety in the parenchyma. Our results are significant on two levels. Firstly, we demonstrated that one can localize all proteins in one analysis and without using laboratory animals (antibodies for immunolocalization of single proteins). Secondly, this study offers the first complex proteomic data on not only the E. nipponicum but within the whole class of Monogenea, which was from this point of view until recently neglected.


Asunto(s)
Mucosa Intestinal/metabolismo , Tejido Parenquimatoso/metabolismo , Platelmintos/metabolismo , Proteoma/análisis , Proteómica/métodos , Animales , Carpas/parasitología , Catepsinas/análisis , Catepsinas/metabolismo , Cromatografía Líquida de Alta Presión , Branquias/parasitología , Captura por Microdisección con Láser , Péptido Hidrolasas/análisis , Péptido Hidrolasas/metabolismo , Espectrometría de Masas en Tándem
12.
Tumour Biol ; 42(6): 1010428320936410, 2020 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-32586207

RESUMEN

Pancreatic ductal adenocarcinoma is the most common and aggressive type of pancreatic cancer, with a 5-year survival rate that is less than 10%. New biomarkers to aid in predicting the prognosis of pancreatic ductal adenocarcinoma patients are needed. Previous proteomic studies have to a great extent focused on finding proteins of value for the diagnosis of pancreatic ductal adenocarcinoma. There is a lack of studies that have profiled the serum or plasma proteome in order to discover candidates for new prognostic biomarkers. In this study, we have used ultra-performance liquid chromatography-ultra-definition mass spectrometry to analyze the serum samples of 21 pancreatic ductal adenocarcinoma patients with short or long survival. Statistical analysis discovered 31 proteins whose expression differed significantly between pancreatic ductal adenocarcinoma patients with short or long survival. Pathway analysis discovered multiple canonical pathways enriched in this data set, with several pathways having roles in inflammation and lipid metabolism. The serum proteins identified here, which include complement components and several enzymes, could be of value as candidates for new noninvasive prognostic markers.


Asunto(s)
Adenocarcinoma/mortalidad , Biomarcadores de Tumor/metabolismo , Proteínas Sanguíneas/metabolismo , Carcinoma Ductal Pancreático/mortalidad , Neoplasias Pancreáticas/mortalidad , Proteoma/metabolismo , Proteómica/métodos , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Anciano , Biomarcadores de Tumor/análisis , Proteínas Sanguíneas/análisis , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proyectos Piloto , Pronóstico , Mapas de Interacción de Proteínas , Proteoma/análisis , Tasa de Supervivencia
13.
PLoS Negl Trop Dis ; 14(6): e0008299, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32511239

RESUMEN

Snake venoms are complex mixtures of proteins with toxic activities, with many distinct isoforms, affecting different physiological targets, comprised in a few protein families. It is currently accepted that this diversity in venom composition is an adaptive advantage for venom efficacy on a wide range of prey. However, on the other side, variability on isoforms expression has implications in the clinics of human victims of snakebites and in the efficacy of antivenoms. B. atrox snakes are responsible for most of the human accidents in Brazilian Amazon and the type and abundance of protein families on their venoms present individual variability. Thus, in this study we attempted to correlate the individual venom proteome of the snake brought to the hospital by the patient seeking for medical assistance with the clinical signs observed in the same patient. Individual variability was confirmed in venoms of the 14 snakes selected for the study. The abundance of each protein family was quite similar among the venom samples, while the isoforms composition was highly variable. Considering the protein families, the SVMP group presented the best correlation with bleeding disorders and edema. Considering individual isoforms, some isoforms of venom metalloproteinase (SVMP), C-type lectin-like toxins (CTL) and snake venom serine proteinases (SVSP) presented expression levels that with statistically significant positive correlation to signs and symptoms presented by the patients as bleeding disorders, edema, ecchymosis and blister formation. However, some unexpected data were also observed as the correlation between a CTL, CRISP or LAAO isoforms with blister formation, still to be confirmed with a larger number of samples. Although this is still a small number of patient samples, we were able to indicate that venom composition modulates clinical manifestations of snakebites, to confirm at the bedside the prominent role of SVMPs and to include new possible toxin candidates for the development of toxin inhibitors or to improve antivenom selectiveness, important actions for the next generation treatments of snakebites.


Asunto(s)
Bothrops , Venenos de Crotálidos/análisis , Proteoma/análisis , Serina Proteasas/análisis , Animales , Antivenenos , Brasil , Metaloproteasas/análisis , Isoformas de Proteínas/análisis , Proteómica , Mordeduras de Serpientes/terapia
14.
Ann Clin Lab Sci ; 50(3): 308-313, 2020 May.
Artículo en Inglés | MEDLINE | ID: mdl-32581017

RESUMEN

OBJECTIVE: The COVID-19 pandemic has challenged the world economically and medically. Understanding and defining the biology of this specific coronavirus infection may lead to targeted therapies to lessen its virulence and expand the host resistance. This study's objective was to apply morphoproteomics to pulmonary lung sections from a forensic autopsy of an untreated COVID-19 victim, so that we may better define its biology from the perspective of its interaction with the host and provide options for therapeutic targets. DESIGN: Morphoproteomic analysis from a case study of this COVID-19 pulmonary infection included immunohistochemical probes to detect phosphorylated p-STAT3 (Tyr 705), as part of the interleukin (IL)-6 pathway; cyclooxygenase (COX)-2, CD8+ cytotoxic lymphocytes, Programmed Death (PD)-1 receptor+ lymphoid cells, CD56+ NK lymphoid cells, CD163+ (M2 polarized monocytes/macrophages), and programmed death-ligand 1 (PD-L1) expression as part of the host response to interaction with the COVID-19 virus. RESULTS: Representative sections of the COVID-19 victim's lung showed: nuclear expression of p-STAT3 (Tyr 705) in many of the alveolar pneumocytes and in occasional endothelial cells; COX-2 expression in the alveolar pneumocytes; a relative paucity of CD8+ cytotoxic lymphocytes; absence of CD56+ NK lymphoid cells; abundance of intra-alveolar and alveolar interstitial CD163+ macrophages/monocytes; PD-L1 expression on occasional macrophages, focally on collections of alveolar pneumocytes, and on cells in the alveolar interstitium; and rare PD-1+ lymphocytes in similar regions as CD8+ lymphocytes. CONCLUSION: Morphoproteomics and microanatomical features coincide with the etiopathogenic features of pulmonary coronavirus infection and the host response. This suggests that a targeted therapy could address the biology of COVID-19 pneumonia, enhance the host immune response and prevent its progression to a life-threatening, ventilator-dependent clinical situation.


Asunto(s)
Betacoronavirus/aislamiento & purificación , Biomarcadores/metabolismo , Infecciones por Coronavirus/complicaciones , Enfermedades Pulmonares/metabolismo , Enfermedades Pulmonares/patología , Neumonía Viral/complicaciones , Proteoma/análisis , Biomarcadores/análisis , Infecciones por Coronavirus/virología , Resultado Fatal , Humanos , Enfermedades Pulmonares/etiología , Masculino , Persona de Mediana Edad , Pandemias , Neumonía Viral/virología , Proteoma/metabolismo
15.
PLoS One ; 15(5): e0232084, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32374735

RESUMEN

Knowledge about the mouse brown adipose tissue (BAT) proteome can provide a deeper understanding of the function of mammalian BAT. Herein, a comprehensive analysis of interscapular BAT from C57BL/6J female mice was conducted by 2DLC and high-resolution mass spectrometry to construct a comprehensive proteome dataset of mouse BAT proteins. A total of 4949 nonredundant proteins were identified, and 4495 were quantified using the iBAQ method. According to the iBAQ values, the BAT proteome was divided into high-, middle- and low-abundance proteins. The functions of the high-abundance proteins were mainly related to glucose and fatty acid oxidation to produce heat for thermoregulation, while the functions of the middle- and low-abundance proteins were mainly related to protein synthesis and apoptosis, respectively. Additionally, 497 proteins were predicted to have signal peptides using SignalP4 software, and 75 were confirmed in previous studies. This study, for the first time, comprehensively profiled and functionally annotated the BAT proteome. This study will be helpful for future studies focused on biomarker identification and BAT molecular mechanisms.


Asunto(s)
Tejido Adiposo Pardo/metabolismo , Anotación de Secuencia Molecular , Proteoma/metabolismo , Proteómica , Tejido Adiposo Pardo/química , Animales , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Bases de Datos de Proteínas , Femenino , Ratones , Ratones Endogámicos C57BL , Anotación de Secuencia Molecular/métodos , Proteoma/análisis , Proteómica/métodos , Espectrometría de Masas en Tándem
16.
PLoS One ; 15(5): e0232553, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32379834

RESUMEN

BACKGROUND: Amniotic fluid is clinically accessible via amniocentesis and its protein composition may correspond to birth timing. Early changes in the amniotic fluid proteome could therefore be associated with the subsequent development of spontaneous preterm delivery. OBJECTIVE: The main objective of this study was to perform unbiased proteomics analysis of the association between mid-trimester amniotic fluid proteome and spontaneous preterm delivery and gestational duration, respectively. A secondary objective was to validate and replicate the findings by enzyme-linked immunosorbent assay using a second independent cohort. METHODS: Women undergoing a mid-trimester genetic amniocentesis at Sahlgrenska University Hospital/Östra between September 2008 and September 2011 were enrolled in this study, designed in three analytical stages; 1) an unbiased proteomic discovery phase using LC-MS analysis of 22 women with subsequent spontaneous preterm delivery (cases) and 37 women who delivered at term (controls), 2) a validation phase of proteins of interest identified in stage 1, and 3) a replication phase of the proteins that passed validation using a second independent cohort consisting of 20 cases and 40 matched controls. RESULTS: Nine proteins were nominally significantly associated with both spontaneous preterm delivery and gestational duration, after adjustment for gestational age at sampling, but none of the proteins were significant after correction for multiple testing. Several of these proteins have previously been described as being associated with spontaneous PTD etiology and six of them were thus validated using enzyme linked immunosorbent assay. Two of the proteins passed validation; Neutrophil gelatinase-associated lipocalin and plasminogen activator inhibitor 1, but the results could not be replicated in a second cohort. CONCLUSIONS: Neutrophil gelatinase-associated lipocalin and Plasminogen activator inhibitor 1 are potential biomarkers of spontaneous preterm delivery and gestational duration but the findings could not be replicated. The negative findings are supported by the fact that none of the nine proteins from the exploratory phase were significant after correction for multiple testing.


Asunto(s)
Líquido Amniótico/metabolismo , Edad Gestacional , Segundo Trimestre del Embarazo/metabolismo , Nacimiento Prematuro/metabolismo , Proteoma/análisis , Adulto , Amniocentesis , Líquido Amniótico/química , Estudios de Cohortes , Femenino , Humanos , Masculino , Embarazo
17.
Food Chem ; 326: 126983, 2020 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-32413763

RESUMEN

Confirmed to be a new type of food resource, quail egg can provide humans with high-quality protein and offer various nutrients that can promote growth and development. Post-translational modification of proteins can regulate their molecular structures and physiological functions. However, the understanding and related research of quail egg holoproteins and post-translationally modified proteins is not yet sufficient. This study provides an in-depth analysis of quail egg proteins using an omics strategy. A total of 175 proteins, 109 N-glycoproteins (293 N-glycosylation sites) and 23 phosphoproteins (84 phosphorylation sites) were identified. Motif analysis showed that N-glycosylation sites of quail eggs were classical sites. The main characteristic sequence of the phosphorylation site is "S-X-E" (77%). Functional analysis indicated that quail egg proteins, modified proteins were enriched in the regulation of enzyme activity. These results have significant reference value for understanding the structure, function of quail eggs, explaining the physicochemical reaction during the storage.


Asunto(s)
Proteínas del Huevo/metabolismo , Huevos/análisis , Codorniz/metabolismo , Espectrometría de Masas en Tándem , Animales , Cromatografía Líquida de Alta Presión , Glicoproteínas/metabolismo , Glicosilación , Humanos , Fosfoproteínas/metabolismo , Fosforilación , Proteoma/análisis
18.
Nat Methods ; 17(5): 505-508, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32371966

RESUMEN

Ligands bound to protein assemblies provide critical information for function, yet are often difficult to capture and define. Here we develop a top-down method, 'nativeomics', unifying 'omics' (lipidomics, proteomics, metabolomics) analysis with native mass spectrometry to identify ligands bound to membrane protein assemblies. By maintaining the link between proteins and ligands, we define the lipidome/metabolome in contact with membrane porins and a mitochondrial translocator to discover potential regulators of protein function.


Asunto(s)
Lípidos/análisis , Espectrometría de Masas/métodos , Proteínas de la Membrana/metabolismo , Metaboloma , Proteoma/análisis , Humanos , Ligandos
19.
Mol Immunol ; 123: 7-17, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32387766

RESUMEN

The identification of T cell epitopes derived from tumour specific antigens remains a significant challenge for the development of peptide-based vaccines and immunotherapies. The use of mass spectrometry-based approaches (immunopeptidomics) can provide powerful new avenues for the identification of such epitopes. In this study we report the use of complementary peptide antigen enrichment methods and a comprehensive mass spectrometric acquisition strategy to provide in-depth immunopeptidome data for the THP-1 cell line, a cell line used widely as a model of human leukaemia. To accomplish this, we combined robust experimental workflows that incorporated ultrafiltration or off-line reversed phase chromatography to enrich peptide ligand as well as a multifaceted data acquisition strategy using an Orbitrap Fusion LC-MS instrument. Using the combined datasets from the two ligand enrichment methods we gained significant depth in immunopeptidome coverage by identifying a total of 41,816 HLA class I peptides from THP-1 cells, including a significant number of peptides derived from different oncogenes or over expressed proteins associated with cancer. The physicochemical properties of the HLA-bound peptides dictated their recovery using the two ligand enrichment approaches and their distribution across the different precursor charge states considered in the data acquisition strategy. The data highlight the complementarity of the two enrichment procedures, and in cases where sample is not limiting, suggest that the combination of both approaches will yield the most comprehensive immunopeptidome information.


Asunto(s)
Antígenos de Neoplasias/análisis , Minería de Datos/métodos , Epítopos de Linfocito T/inmunología , Leucemia Mieloide Aguda/metabolismo , Péptidos/análisis , Proteoma/análisis , Antígenos de Neoplasias/química , Antígenos de Neoplasias/inmunología , Células Cultivadas , Cromatografía Liquida/métodos , Bases de Datos de Proteínas , Humanos , Inmunoterapia/métodos , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/terapia , Ligandos , Espectrometría de Masas/métodos , Biblioteca de Péptidos , Péptidos/química , Proteoma/química , Proteómica/métodos , Células THP-1
20.
Proc Natl Acad Sci U S A ; 117(22): 12109-12120, 2020 06 02.
Artículo en Inglés | MEDLINE | ID: mdl-32414919

RESUMEN

The mitochondria-associated membrane (MAM) has emerged as a cellular signaling hub regulating various cellular processes. However, its molecular components remain unclear owing to lack of reliable methods to purify the intact MAM proteome in a physiological context. Here, we introduce Contact-ID, a split-pair system of BioID with strong activity, for identification of the MAM proteome in live cells. Contact-ID specifically labeled proteins proximal to the contact sites of the endoplasmic reticulum (ER) and mitochondria, and thereby identified 115 MAM-specific proteins. The identified MAM proteins were largely annotated with the outer mitochondrial membrane (OMM) and ER membrane proteins with MAM-related functions: e.g., FKBP8, an OMM protein, facilitated MAM formation and local calcium transport at the MAM. Furthermore, the definitive identification of biotinylation sites revealed membrane topologies of 85 integral membrane proteins. Contact-ID revealed regulatory proteins for MAM formation and could be reliably utilized to profile the proteome at any organelle-membrane contact sites in live cells.


Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Proteoma/análisis , Proteínas de Unión a Tacrolimus/metabolismo , Calcio/metabolismo , Humanos , Biogénesis de Organelos , Proteoma/metabolismo , Transducción de Señal
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