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Tolfenpyrad (TFP) is an extensively used pesticide that inevitably leads to human exposure to both TFP and its transformation product residues. However, the biotransformation of TFP in humans has not been elucidated, and the toxicity of TFP along with its biotransformation products remains largely unknown. In this study, the biotransformation process of TFP was investigated using human liver microsomes and human hepatic cells. Endogenous metabolic changes in the cells were studied to investigate the hepatocytotoxicity of TFP at environmentally relevant concentrations. Fourteen phase I biotransformation products and four phase II TFP products were characterized, among which twelve products were identified for the first time. The oxidative product tolfenpyrad-benzoic acid (PT-CA) was particularly abundant and stable. Further hepatotoxicity assessments and metabolic studies demonstrated comparable metabolic profiles for TFP and PT-CA in HepG2 cells, with both significantly disrupting purine and glutathione metabolism. These processes are closely associated with oxidative stress, mitochondrial damage, and cell death. Our results provide novel perspectives on the biotransformation, metabolism, and hepatotoxicity of TFP, thereby highlighting the non-negligible toxicity of its crucial biotransformation product PT-CA in environmental risk assessments.
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The difficulty of short-process bonded Nd-Fe-B magnet waste recycling lies in the effective removal of the cured polymer matrix while protecting the magnetic powder. In this study, the polymer matrix in bonded Nd-Fe-B magnet waste was destroyed using sodium hydroxide ethanol solution, and the effect of the recycling process on the magnetic powders was studied. The nonmagnetic polymer matrix was removed, while the magnetic phase was not destroyed. The carbon and oxygen contents of the recycled magnetic powders decreased by 92.96 and 89.30%, respectively, while the MS (saturation magnetization), Mr (remanence), and Hcj (coercivity) values of the recycled magnetic powders were 99.8, 98.5, and 95.9% of the original magnetic powders, respectively. The curing and decomposition processes of the polymer matrix were also analyzed. During the curing process, dicyandiamide and bisphenol A epoxy resin acted as bridges and skeletons, respectively, finally forming a thermosetting three-dimensional network structure. In the alkaline alcohol solution, the bridges and skeletons were destroyed by the free hydroxyl groups and free hydrogen radicals in ethanol, and small molecular products were dissolved in the solution.
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The Empirical Statistical Model (ESM) for decoy library searching fused the expected amino acid sequence of 18 non-human protein standards to a human decoy library. The ESM assumed a priori the standards were pure such that only the 18 nominal proteins were true positive, all other proteins were false positive, there was no overlap in the peptides of non-human proteins versus human proteins, and that the score distribution of individual peptides would resolve true positive from false positive results or noise. The results of random and independent sampling by LC-ESI-MS/MS indicated that the fundamental assumptions of the ESM were not in good agreement with the actual purity of the commercial test standards and so the method showed a 99.7% false negative rate. The ESM for decoy library searching apparently showed poor agreement with SDS-PAGE using silver staining, goodness of fit of MS/MS spectra by X!TANDEM, FDR correction by Benjamini and Hochberg, or comparison to the observation frequency of null random MS/MS spectra, that all confirmed the standards contain hundreds of proteins with a low FDR of primary structural identification. The protein observation frequency increased with abundance and the log10 precursor intensity distributions were Gaussian and nearly ideal for relative quantification.
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Bases de Datos de Proteínas , Proteínas/normas , Animales , Humanos , Estándares de Referencia , Espectrometría de Masas en TándemRESUMEN
A Rabbit myosin standard, like that used to create the empirical statistical model, was randomly and independently sampled by liquid chromatography micro electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) with a linear quadrupole ion trap. The rabbit myosin protein standard appeared pure by SDS-PAGE and CBBR staining but showed many other proteins by silver staining. The LC-MS intensity from myosin and IgG samples were above the 99% safe limit of detection and quantification computed from 36 blank LC-ESI-MS/MS runs. The myosin contained ≤406 Gene Symbols, open reading frames or loci where 79 protein types showed ≥3 peptides from X!TANDEM. Myosins, actin, troponin, other proteins showed 95%-100% homology between the rabbit versus the human decoy library. The myosin protein complex from STRING was true positive compared to random or noise spectra MS/MS with a low type I error (p-value) and low FDR (q-value) computed in R. SDS-PAGE, Western blot, comparison to random and noise MS/MS spectra, X!TANDEM p-values, FDR corrected q-values, and STRING all agreed that the error rate of LC-ESI-MS/MS with a quadrupole ion trap is far below that assumed a priori by the design of the empirical statistical model for decoy library searching.
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Miosinas/química , Miosinas/normas , Animales , Cromatografía Liquida/métodos , Inmunoglobulina G/química , Modelos Estadísticos , Péptidos/química , Conejos , Estándares de Referencia , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
We introduce THRIVE (Tumor Heterogeneity Research Interactive Visualization Environment), an open-source tool developed to assist cancer researchers in interactive hypothesis testing. The focus of this tool is to quantify spatial intratumoral heterogeneity (ITH), and the interactions between different cell phenotypes and noncellular constituents. Specifically, we foresee applications in phenotyping cells within tumor microenvironments, recognizing tumor boundaries, identifying degrees of immune infiltration and epithelial/stromal separation, and identification of heterotypic signaling networks underlying microdomains. The THRIVE platform provides an integrated workflow for analyzing whole-slide immunofluorescence images and tissue microarrays, including algorithms for segmentation, quantification, and heterogeneity analysis. THRIVE promotes flexible deployment, a maintainable code base using open-source libraries, and an extensible framework for customizing algorithms with ease. THRIVE was designed with highly multiplexed immunofluorescence images in mind, and, by providing a platform to efficiently analyze high-dimensional immunofluorescence signals, we hope to advance these data toward mainstream adoption in cancer research. Cancer Res; 77(21); e71-74. ©2017 AACR.
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Heterogeneidad Genética , Neoplasias/genética , Imagen Óptica/estadística & datos numéricos , Programas Informáticos , Algoritmos , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Neoplasias/patología , Imagen Óptica/métodos , Análisis de Matrices Tisulares/estadística & datos numéricosRESUMEN
It will be important to determine if the parent and fragment ion intensity results of liquid chromatography, electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) experiments have been randomly and independently sampled from a normal population for the purpose of statistical analysis by general linear models and ANOVA. The tryptic parent peptide and fragment ion m/z and intensity data in the mascot generic files from LC-ESI-MS/MS of purified standard proteins, and human blood protein fractionated by partition chromatography, were parsed into a Structured Query Language (SQL) database and were matched with protein and peptide sequences provided by the X!TANDEM algorithm. The many parent and/or fragment ion intensity values were log transformed, tested for normality, and analyzed using the generic Statistical Analysis System (SAS). Transformation of both parent and fragment intensity values by logarithmic functions yielded intensity distributions that closely approximate the log-normal distribution. ANOVA models of the transformed parent and fragment intensity values showed significant effects of treatments, proteins, and peptides, as well as parent versus fragment ion types, with a low probability of false positive results. Transformed parent and fragment intensity values were compared over all sample treatments, proteins or peptides by the Tukey-Kramer Honestly Significant Difference (HSD) test. The approach provided a complete and quantitative statistical analysis of LC-ESI-MS/MS data from human blood.
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Proteínas Sanguíneas/análisis , Cromatografía Liquida/métodos , Proteómica/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Cromatografía Liquida/estadística & datos numéricos , Humanos , Proteómica/estadística & datos numéricos , Espectrometría de Masa por Ionización de Electrospray/estadística & datos numéricos , Estadística como Asunto , Espectrometría de Masas en TándemRESUMEN
This paper proposes a novel formulation to model and analyze the statistical characteristics of some types of segmentation problems that are based on combining label maps / templates / atlases. Such segmentation-by-example approaches are quite powerful on their own for several clinical applications and they provide prior information, through spatial context, when combined with intensity-based segmentation methods. The proposed formulation models a class of multiatlas segmentation problems as nonparametric regression problems in the high-dimensional space of images. The paper presents a systematic analysis of the nonparametric estimation's convergence behavior (i.e. characterizing segmentation error as a function of the size of the multiatlas database) and shows that it has a specific analytic form involving several parameters that are fundamental to the specific segmentation problem (i.e. chosen anatomical structure, imaging modality, registration method, label-fusion algorithm, etc.). We describe how to estimate these parameters and show that several brain anatomical structures exhibit the trends determined analytically. The proposed framework also provides per-voxel confidence measures for the segmentation. We show that the segmentation error for large database sizes can be predicted using small-sized databases. Thus, small databases can be exploited to predict the database sizes required ("how many templates") to achieve "good" segmentations having errors lower than a specified tolerance. Such cost-benefit analysis is crucial for designing and deploying multiatlas segmentation systems.
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This paper presents a fast method for quantifying shape differences/similarities between pairs of magnetic resonance (MR) brain images. Most shape comparisons in the literature require some kind of deformable registration or identification of exact correspondences. The proposed approach relies on an optimal matching of a large collection of features, using a very fast, hierarchical method from the literature, called spatial pyramid matching (SPM). This paper shows that edge-based image features in combination with SPM results in a fast similarity measure that captures relevant anatomical information in brain MRI. We present extensive comparisons against known methods for shape-based, k-nearest-neighbor lookup to evaluate the performance of the proposed method. Finally, we show that the method compares favorably with more computation-intensive methods in the construction of local atlases for use in brain MR image segmentation.
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Encéfalo/patología , Procesamiento de Imagen Asistido por Computador/métodos , Imagen por Resonancia Magnética/métodos , Algoritmos , Mapeo Encefálico/métodos , Análisis por Conglomerados , Simulación por Computador , Humanos , Modelos Estadísticos , Red Nerviosa , Reconocimiento de Normas Patrones Automatizadas/métodos , Técnica de SustracciónRESUMEN
Retinal ganglion cells (RGCs) are central nervous system (CNS) neurons that transmit visual information from the retina to the brain. Apoptotic RGC degeneration causes visual impairment that can be modeled by optic nerve crush. Neuronal apoptosis is also a salient feature of CNS trauma, ischemia (stroke), and diseases of the CNS such as Alzheimer's, Parkinson's, multiple sclerosis, and amyotrophic lateral sclerosis. Optic nerve crush induces the apoptotic cell death of â¼ 70% of RGCs within the first 14 days after injury. This model is particularly attractive for studying adult neuron apoptosis because the time-course of RGC death is well established and axon regeneration within the myelinated optic nerve can be concurrently evaluated. Here, we performed a large scale iTRAQ proteomic study to identify and quantify proteins of the rat retina at 1, 3, 4, 7, 14, and 21 days after optic nerve crush. In total, 337 proteins were identified, and 110 were differentially regulated after injury. Of these, 58 proteins were upregulated (>1.3 ×), 46 were downregulated (<0.7 ×), and 6 showed both positive and negative regulation over 21 days, relative to normal retinas. Among the differentially expressed proteins, Thymosin-ß4 showed an early upregulation at 3 days, the time-point that immediately precedes the induction of RGC apoptosis after injury. We examined the effect of exogenous Thymosin-ß4 administration on RGC death after optic nerve injury. Intraocular injections of Thymosin-ß4 significantly increased RGC survival by â¼ 3-fold compared to controls and enhanced axon regeneration after crush, demonstrating therapeutic potential for CNS insults. Overall, our study identified numerous proteins that are differentially regulated at key time-points after optic nerve crush, and how the temporal profiles of their expression parallel RGC death. This data will aid in the future development of novel therapeutics to promote neuronal survival and regeneration in the adult CNS.
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Traumatismos del Nervio Óptico/patología , Células Ganglionares de la Retina/patología , Espectrometría de Masas en Tándem/métodos , Animales , Apoptosis , Cromatografía Líquida de Alta Presión , Proteínas del Ojo/metabolismo , Traumatismos del Nervio Óptico/metabolismo , Proteómica , Ratas , Células Ganglionares de la Retina/metabolismoRESUMEN
Myogenesis is a well-characterized program of cellular differentiation that is exquisitely sensitive to the extracellular milieu. Systematic characterization of the myogenic secretome (i.e. the ensemble of secreted proteins) is, therefore, warranted for the identification of novel secretome components that regulate both the pluripotency of these progenitor mesenchymal cells, and also their commitment and passage through the differentiation program. Previously, we have successfully identified 26 secreted proteins in the mouse skeletal muscle cell line C2C12 (1). In an effort to attain a more comprehensive picture of the regulation of myogenesis by its extracellular milieu, quantitative profiling employing stable isotope labeling by amino acids in cell culture was implemented in conjunction with two parallel high throughput online reverse phase liquid chromatography-tandem mass spectrometry systems. In summary, 34 secreted proteins were quantified, 30 of which were shown to be differentially expressed during muscle development. Intriguingly, our analysis has revealed several novel up- and down-regulated secretome components that may have critical biological relevance for both the maintenance of pluripotency and the passage of cells through the differentiation program. In particular, the altered regulation of secretome components, including follistatin-like protein-1, osteoglycin, spondin-2, and cytokine-induced apoptosis inhibitor-1, along with constitutively expressed factors, such as fibulin-2, illustrate dynamic changes in the secretome that take place when differentiation to a specific lineage occurs.
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Desarrollo de Músculos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/fisiología , Mioblastos Esqueléticos/metabolismo , Proteoma/metabolismo , Secuencia de Aminoácidos , Animales , Isótopos de Carbono , Técnicas de Cultivo de Célula , Diferenciación Celular , Medios de Cultivo Condicionados/análisis , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Marcaje Isotópico , Luciferasas/biosíntesis , Luciferasas/genética , Ratones , Datos de Secuencia Molecular , Fibras Musculares Esqueléticas/citología , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/citología , Fragmentos de Péptidos/química , Regiones Promotoras Genéticas , Proteoma/química , Espectrometría de Masas en TándemRESUMEN
The simplest model-that authentic tandem mass spectrometry (MS/MS) spectra are no different from noise, random spectra, or false-positive results-may be directly examined by chi-square comparison of the peptide-to-protein distribution. The peptide-to-protein distribution of a set of 4151 redundant blood proteins identified by X!TANDEM indicated that there is a low probability that the authentic data were the same as noise, random spectra, or false-positive correlations (P<0.0001). In contrast, a competition for significance failed to distinguish approximately 90% of authentic blood proteins from those of noise, random spectra, or false-positive results (P<0.01) and apparently incurred a large type II error (false negative). The chi-square test of peptide-to-protein frequency distributions was found to be an efficient means to distinguish authentic data from false-positive results. Frequency-based statistics unambiguously demonstrated that proteins can be identified by liquid chromatography-electrospray ionization-MS/MS from human blood with acceptable confidence. Thus, the chi-square fit of the peptide-to-protein distribution could distinguish authentic data from random or false-positive data, but the score distribution method could not separate real results from false results.
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Proteínas Sanguíneas/química , Cromatografía Líquida de Alta Presión/métodos , Péptidos/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Algoritmos , Secuencia de Aminoácidos , Animales , Bases de Datos de Proteínas , Humanos , Datos de Secuencia Molecular , Pez CebraRESUMEN
Proteomics uses tandem mass spectrometers and correlation algorithms to match peptides and their fragment spectra to amino acid sequences. The replication of multiple liquid chromatography experiments with electrospray ionization of peptides and tandem mass spectrometry (LC-ESI-MS/MS) produces large sets of MS/MS spectra. There is a need to assess the quality of large sets of experimental results by statistical comparison with that of random expectation. Classical frequency-based statistics such as goodness-of-fit tests for peptide-to-protein distributions could be used to calculate the probability that an entire set of experimental results has arisen by random chance. The frequency distributions of authentic MS/MS spectra from human blood were compared with those of false positive MS/MS spectra generated by a computer, or instrument noise, using the chi-square test. Here the mechanics of the chi-square test to compare the results in toto from a set of LC-ESI-MS/MS experiments with those of random expectation is detailed. The chi-square analysis of authentic spectra demonstrates unambiguously that the analysis of blood proteins separated by partition chromatography prior to tryptic digestions has a low probability that the cumulative peptide-to-protein distribution is the same as that of random or noise false positive spectra.
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Proteínas Sanguíneas/análisis , Péptidos/análisis , Proteínas/análisis , Espectrometría de Masas en Tándem/métodos , Algoritmos , Cromatografía Liquida , Reacciones Falso Positivas , Humanos , Proteoma/análisis , Proteómica/métodos , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
It is difficult to convey the accelerating rate and growing importance of mass spectrometry applications to human blood proteins and peptides. Mass spectrometry can rapidly detect and identify the ionizable peptides from the proteins in a simple mixture and reveal many of their post-translational modifications. However, blood is a complex mixture that may contain many proteins first expressed in cells and tissues. The complete analysis of blood proteins is a daunting task that will rely on a wide range of disciplines from physics, chemistry, biochemistry, genetics, electromagnetic instrumentation, mathematics and computation. Therefore the comprehensive discovery and analysis of blood proteins will rank among the great technical challenges and require the cumulative sum of many of mankind's scientific achievements together. A variety of methods have been used to fractionate, analyze and identify proteins from blood, each yielding a small piece of the whole and throwing the great size of the task into sharp relief. The approaches attempted to date clearly indicate that enumerating the proteins and peptides of blood can be accomplished. There is no doubt that the mass spectrometry of blood will be crucial to the discovery and analysis of proteins, enzyme activities, and post-translational processes that underlay the mechanisms of disease. At present both discovery and quantification of proteins from blood are commonly reaching sensitivities of â¼1 ng/mL.
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Proteínas Sanguíneas/aislamiento & purificación , Espectrometría de Masas/métodos , Procesamiento Proteico-Postraduccional , Proteínas Sanguíneas/metabolismo , Bases de Datos de Proteínas , Endopeptidasas/química , Humanos , Espectrometría de Masas/instrumentación , Proteolisis , Estándares de Referencia , Sensibilidad y Especificidad , Manejo de EspecímenesRESUMEN
In this paper we present a new method for spatial regularization of functional connectivity maps based on Markov Random Field (MRF) priors. The high level of noise in fMRI leads to errors in functional connectivity detection algorithms. A common approach to mitigate the effects of noise is to apply spatial Gaussian smoothing, which can lead to blurring of regions beyond their actual boundaries and the loss of small connectivity regions. Recent work has suggested MRFs as an alternative spatial regularization in detection of fMRI activation in task-based paradigms. We propose to apply MRF priors to the computation of functional connectivity in resting-state fMRI. Our Markov priors are in the space of pairwise voxel connections, rather than in the original image space, resulting in a MRF whose dimension is twice that of the original image. The high dimensionality of the MRF estimation problem leads to computational challenges. We present an efficient, highly parallelized algorithm on the Graphics Processing Unit (GPU). We validate our approach on a synthetically generated example as well as real data from a resting state fMRI study.
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Algoritmos , Mapeo Encefálico/métodos , Encéfalo/fisiología , Potenciales Evocados/fisiología , Interpretación de Imagen Asistida por Computador/métodos , Imagen por Resonancia Magnética/métodos , Vías Nerviosas/fisiología , Interpretación Estadística de Datos , Humanos , Aumento de la Imagen/métodos , Cadenas de Markov , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Sequence analysis of the blood peptides and their qualities will be key to understanding the mechanisms that contribute to error in LC-ESI-MS/MS. Analysis of peptides and their proteins at the level of sequences is much more direct and informative than the comparison of disparate accession numbers. A portable database of all blood peptide and protein sequences with descriptor fields and gene ontology terms might be useful for designing immunological or MRM assays from human blood. The results of twelve studies of human blood peptides and/or proteins identified by LC-MS/MS and correlated against a disparate array of genetic libraries were parsed and matched to proteins from the human ENSEMBL, SwissProt and RefSeq databases by SQL. The reported peptide and protein sequences were organized into an SQL database with full protein sequences and up to five unique peptides in order of prevalence along with the peptide count for each protein. Structured query language or BLAST was used to acquire descriptive information in current databases. Sampling error at the level of peptides is the largest source of disparity between groups. Chi Square analysis of peptide to protein distributions confirmed the significant agreement between groups on identified proteins.
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Proteínas Sanguíneas/química , Proteómica/métodos , Algoritmos , Cromatografía Liquida/métodos , Reacciones Falso Negativas , Reacciones Falso Positivas , Humanos , Modelos Estadísticos , Péptidos/química , Proteínas/química , Proteoma , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Tripsina/químicaRESUMEN
The endogenous peptides of human serum may have regulatory functions, have been associated with physiological states, and their modifications may reveal some mechanisms of disease. In order to correlate levels of specific peptides with disease alongside internal standards, the polypeptides must first be reliably extracted and identified. Endogenous blood peptides can be effectively enriched by precipitation of the serum with organic solvents followed by selective extraction of peptides using aqueous solutions modified with organic solvents. Polypeptides on filter paper were assayed with Coomasie brilliant blue binding. The polypeptides were resolved by detergent tricine polyacrylamide electrophoresis and visualized by diamine silver staining. Peptides in the extracts were collected by C18 and analyzed by matrix-assisted laser desorption/ionization and liquid chromatography-electrospray ionization-tandem mass spectrometry (MS/MS) quadrupole time-of-flight MS/MS. Peptides were resolved as multiple isotopic peaks in MS mode with mass deviation of 0.1 Da or less and similar accuracy for fragments. The sensitivity of MS and MS/MS analysis was estimated to be in the picomolar range or less. The peptide composition of the extracts was dependent on solvent formulation. Multiple peptides from apolipoproteins, complement proteins, coagulation factors, and many others were identified by X!Tandem with high mass accuracy of peptide ions and fragments from collision-induced dissociation. Many previously unreported posttranslational modifications of peptides including phosphorylations, oxidations, glycosylations, and others were detected with high mass accuracy and may be of clinical importance. About 4,630 redundant peptides were identified with 99% confidence separately, and together some 1,251 distinct proteins were identified with 99% confidence or greater using the Paragon algorithm.
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Péptidos/sangre , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Aminoácidos , Precipitación Química , Electroforesis en Gel de Poliacrilamida , Humanos , Datos de Secuencia Molecular , Péptidos/análisis , Sensibilidad y Especificidad , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
The roles of inositol-1,4,5-trisphosphate receptors (IP3Rs) in arrhythmia are not fully understood, especially in human beings. Recently, the reported upregulated expression of IP3Rs in atrial myocytes of atrial fibrillation (AF) subjects suggested that IP3Rs might be associated with AF. To directly understand the roles of IP3Rs in AF, we have investigated the IP3R-dependent Ca2+ events as well as the cross-talk between IP3Rs and ryanodine receptors (RyRs) in permeabilized atrial myocytes of AF and normal sinus rhythm (NSR) patients by Ca2+ imaging. In the presence of tetracaine, IP(3)R-dependent Ca2+ events in AF atrial myocytes showed increased frequency, delayed termination and broadened width, compared with NSR myocytes. Moreover, when RyRs were not inhibited, IP3 or adenophostin induced an outburst of RyR-dependent spontaneous Ca2+ sparks with the altered spatial-temporal characteristics. The activation of IP3Rs also enhanced Ca2+ waves. These effects on RyR-dependent Ca2+ signaling were significantly stronger in AF myocytes than in NSR cells and were completely blocked by 2-aminoethoxydiphenyl borate. Thus, our results suggested not only an enhanced activity of IP3Rs but also an elevated cross-talk between IP3R- and RyR-mediated Ca2+ signaling in atrial myocytes of human AF patients, a reflection of altered function of IP3Rs in AF.
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Fibrilación Atrial/metabolismo , Señalización del Calcio , Calcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Miocitos Cardíacos/metabolismo , Estudios de Casos y Controles , Permeabilidad de la Membrana Celular , Femenino , Atrios Cardíacos/citología , Atrios Cardíacos/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/agonistas , Masculino , Persona de Mediana Edad , Receptor Cross-Talk , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
Many proteomics studies are limited to the identification of only the most abundant proteins in a sample due to the high sample complexity in most proteomes. We have here addressed this problem by prefractionation of human blood samples using microchromatography. We show that our approach resulted in high-stringency tryptic peptides identified by LC-ESI-MS/MS. Serum proteins were fractionated by batch and stepwise preparative chromatography using various types of chromatography resins (propyl sulfate, quaternary amine, diethylaminoethanol, cibachron blue, phenol Sepharose, carboxy methyl sepharose, hydroxyl apatite, heparin, concanavalin A and protein G) that were compared. The efficacy of sample fractionation was determined by protein assays, electrophoresis, and mass spectrometry. Tryptic peptides were separated by C18 liquid chromatography with electrospray ionization via metal needle at 2 microL/min with ion trap tandem mass spectrometry. The MS/MS spectra were correlated to some 4396 distinct sequences of the human forward RefSeq by X!TANDEM. Of these, 61% have been detected by other algorithms, but 3219 (73%) were never previously reported from blood by X!TANDEM. The use of a simple apparatus for making gravity microchromatography columns that permits the rapid side-by-side fractionation of many serum samples is described. Disposable microcolumns rapidly prepared blood samples for LC ESI-MS/MS that detected both tissue and cell leakage proteins known to exist in the approximately 1 ng/mL range and some circulating receptor sequences. Our results demonstrate that the depletion of albumin or IgG was not necessary prior to LC-MS/MS and that multiple forms of protein chromatography will be useful for complete identification of blood proteins.
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Proteínas Sanguíneas/análisis , Cromatografía Liquida/métodos , Proteoma/metabolismo , Proteínas Sanguíneas/aislamiento & purificación , Humanos , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
Cell surface receptors and their associated signaling pathways on the plasma membrane are key targets in understanding cellular responses. However, the isolation and identification of receptor complexes has been elusive. The Fc receptor was captured from the surface of live cells using microbeads coated with the receptor's cognate ligand, gamma globulin (IgG), and analyzed by liquid chromatography and tandem mass spectrometry (LC-MS/MS) alongside several controls. Live-cell affinity receptor chromatography (LARC) resulted in a partially nonredundant list of 288 proteins that were specific to the Fc receptor complex. The proteins identified were in close agreement with previously determined factors in the Fc receptor complex as demonstrated by genetic and biochemical methods and revealed novel complex members. Confocal microscopy was used to confirm recruitment of SRC, SYK, PLC, PKC, PI3K, SHIP, TEC, CDC42, RAP, PAK, GAP, GEF, GRP, and CRK to the receptor complex upon activation by the same ligand microbeads. The expression of mutants and silencing RNA against specific isoforms were used to demonstrate a functional role for novel members of the Fc receptor complex, including RHOG (RAS homologue member G), p115 RhoGEF (protein of 115-kDa RAS homologue guanine exchange factor), and CRKL (CRK-like). The recruitment of AKT pleckstrin homology (PH) domain green fluorescent protein (GFP) was used to quantify the production of phosphorylated inositol at the activated receptor complex. We conclude that it is feasible to capture an activated receptor complex from the surface of live cells using ligand-coated microbeads for identification of members of a receptor complex or pathway by LC-MS/MS.
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Cromatografía de Afinidad/métodos , Receptores Fc/química , Animales , Sitios de Unión , Línea Celular , Humanos , Inmunoglobulina G/química , Macrófagos/química , Ratones , Microscopía Confocal , Microesferas , Proteínas Proto-Oncogénicas c-akt/análisis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Fc/análisis , Receptores Fc/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
BACKGROUND: Many alterations in sarcoplasmic reticulum Ca(2+) handling proteins in atrial myocytes have been associated with atrial fibrillation (AF) in clinical patients, whereas the functional consequences of these alterations mostly remain unclear. METHODS AND RESULTS: To know whether or not ryanodine receptor (RyR)-mediated intracellular Ca(2+) events in AF atrial myocytes are affected by protein alterations, we investigated spontaneous Ca(2+) sparks and Ca(2+) waves in intact and permeabilized atrial myocytes of AF patients (n(patients) = 21) and normal sinus rhythm (NSR) patients (n(patients) = 22) by laser scanning confocal microscopy. It was found that the frequency, amplitude and rise time of Ca(2+) sparks were comparable between AF and NSR groups, while full width and full duration at half maximum intensity significantly increased in the AF group. Along with these changes, the frequency of small and global Ca(2+) waves increased in AF atrial myocytes. CONCLUSIONS: Our results clearly indicated that the spatiotemporal properties but not the frequency of Ca(2+) sparks were affected in AF atrial myocytes. In addition, the frequency of Ca(2+) waves increased. This profile of the alterations in RyR-mediated Ca(2+) events in AF atrial myocytes was different from previous studies. The underlying mechanisms, as well as possible reasons for this discrepancy, were discussed.