RESUMEN
Over the past decade, a suite of new mass-spectrometry-based proteomics methods has been developed that now enables the conformational properties of proteins and protein-ligand complexes to be studied in complex biological mixtures, from cell lysates to intact cells. Highlighted here are seven of the techniques in this new toolbox. These techniques include chemical cross-linking (XL-MS), hydroxyl radical footprinting (HRF), Drug Affinity Responsive Target Stability (DARTS), Limited Proteolysis (LiP), Pulse Proteolysis (PP), Stability of Proteins from Rates of Oxidation (SPROX), and Thermal Proteome Profiling (TPP). The above techniques all rely on conventional bottom-up proteomics strategies for peptide sequencing and protein identification. However, they have required the development of unconventional proteomic data analysis strategies. Discussed here are the current technical challenges associated with these different data analysis strategies as well as the relative analytical capabilities of the different techniques. The new biophysical capabilities that the above techniques bring to bear on proteomic research are also highlighted in the context of several different application areas in which these techniques have been used, including the study of protein ligand binding interactions (e.g., protein target discovery studies and protein interaction network analyses) and the characterization of biological states.
Asunto(s)
Espectrometría de Masas/métodos , Procesamiento Proteico-Postraduccional , Proteínas/química , Proteoma/química , Proteómica/tendencias , Animales , Reactivos de Enlaces Cruzados/química , Bases de Datos de Proteínas , Medición de Intercambio de Deuterio/métodos , Humanos , Marcaje Isotópico/métodos , Ligandos , Espectrometría de Masas/instrumentación , Unión Proteica , Pliegue de Proteína , Estabilidad Proteica , Proteínas/metabolismo , Proteínas/ultraestructura , Proteolisis , Proteoma/ultraestructura , Proteómica/instrumentación , Proteómica/métodos , Análisis de Secuencia de Proteína/instrumentación , Análisis de Secuencia de Proteína/métodos , Análisis de Secuencia de Proteína/estadística & datos numéricos , TermodinámicaRESUMEN
Proteomics techniques can identify thousands of phosphorylation sites in a single experiment, the majority of which are new and lack precise information about function or molecular mechanism. Here we present a fast method to predict potential phosphorylation switches by mapping phosphorylation sites to protein-protein interactions of known structure and analysing the properties of the protein interface. We predict 1024 sites that could potentially enable or disable particular interactions. We tested a selection of these switches and showed that phosphomimetic mutations indeed affect interactions. We estimate that there are likely thousands of phosphorylation mediated switches yet to be uncovered, even among existing phosphorylation datasets. The results suggest that phosphorylation sites on globular, as distinct from disordered, parts of the proteome frequently function as switches, which might be one of the ancient roles for kinase phosphorylation.
Asunto(s)
Modelos Químicos , Fosfotransferasas/química , Mapeo de Interacción de Proteínas/métodos , Proteoma/química , Análisis de Secuencia de Proteína/métodos , Sitios de Unión , Simulación por Computador , Modelos Moleculares , Fosforilación , Fosfotransferasas/ultraestructura , Unión Proteica , Conformación Proteica , Proteoma/ultraestructura , Relación Estructura-ActividadRESUMEN
The proteome of human brain synapses is highly complex and is mutated in over 130 diseases. This complexity arose from two whole-genome duplications early in the vertebrate lineage. Zebrafish are used in modelling human diseases; however, its synapse proteome is uncharacterized, and whether the teleost-specific genome duplication (TSGD) influenced complexity is unknown. We report the characterization of the proteomes and ultrastructure of central synapses in zebrafish and analyse the importance of the TSGD. While the TSGD increases overall synapse proteome complexity, the postsynaptic density (PSD) proteome of zebrafish has lower complexity than mammals. A highly conserved set of â¼1,000 proteins is shared across vertebrates. PSD ultrastructural features are also conserved. Lineage-specific proteome differences indicate that vertebrate species evolved distinct synapse types and functions. The data sets are a resource for a wide range of studies and have important implications for the use of zebrafish in modelling human synaptic diseases.
Asunto(s)
Encéfalo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteoma/metabolismo , Proteoma/ultraestructura , Sinapsis/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Encéfalo/ultraestructura , Femenino , Duplicación de Gen , Genoma , Masculino , Ratones , Microscopía Electrónica de Transmisión , Modelos Biológicos , Proteínas del Tejido Nervioso/genética , Densidad Postsináptica/metabolismo , Proteoma/genética , Especificidad de la Especie , Sinapsis/ultraestructura , Sinaptosomas/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
The biology of multicellular organisms is coordinated across multiple size scales, from the subnanoscale of molecules to the macroscale, tissue-wide interconnectivity of cell populations. Here we introduce a method for super-resolution imaging of the multiscale organization of intact tissues. The method, called magnified analysis of the proteome (MAP), linearly expands entire organs fourfold while preserving their overall architecture and three-dimensional proteome organization. MAP is based on the observation that preventing crosslinking within and between endogenous proteins during hydrogel-tissue hybridization allows for natural expansion upon protein denaturation and dissociation. The expanded tissue preserves its protein content, its fine subcellular details, and its organ-scale intercellular connectivity. We use off-the-shelf antibodies for multiple rounds of immunolabeling and imaging of a tissue's magnified proteome, and our experiments demonstrate a success rate of 82% (100/122 antibodies tested). We show that specimen size can be reversibly modulated to image both inter-regional connections and fine synaptic architectures in the mouse brain.
Asunto(s)
Encéfalo/metabolismo , Imagenología Tridimensional/métodos , Imagen Molecular/métodos , Proteoma/metabolismo , Sinapsis/metabolismo , Sinapsis/ultraestructura , Animales , Encéfalo/ultraestructura , Femenino , Perfilación de la Expresión Génica/métodos , Aumento de la Imagen/métodos , Interpretación de Imagen Asistida por Computador/métodos , Inmunoensayo/métodos , Masculino , Ratones , Proteínas del Tejido Nervioso/metabolismo , Proteoma/ultraestructura , Distribución TisularRESUMEN
The inbred strains C57BL/6J and DBA/2J (DBA) display striking differences in a number of behavioral tasks depending on hippocampal function, such as contextual memory. Historically, this has been explained through differences in postsynaptic protein expression underlying synaptic transmission and plasticity. We measured the synaptic hippocampal protein content (iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) and mass spectrometry), CA1 synapse ultrastructural morphology, and synaptic functioning in adult C57BL/6J and DBA mice. DBA mice showed a prominent decrease in the Ras-GAP calcium-sensing protein RASAL1. Furthermore, expression of several presynaptic markers involved in exocytosis, such as syntaxin (Stx1b), Ras-related proteins (Rab3a/c), and rabphilin (Rph3a), was reduced. Ultrastructural analysis of CA1 hippocampal synapses showed a significantly lower number of synaptic vesicles and presynaptic cluster size in DBA mice, without changes in postsynaptic density or active zone. In line with this compromised presynaptic morphological and molecular phenotype in DBA mice, we found significantly lower paired-pulse facilitation and enhanced short term depression of glutamatergic synapses, indicating a difference in transmitter release and/or refilling mechanisms. Taken together, our data suggest that in addition to strain-specific postsynaptic differences, the change in dynamic properties of presynaptic transmitter release may underlie compromised synaptic processing related to cognitive functioning in DBA mice.
Asunto(s)
Cognición/fisiología , Hipocampo , Memoria/fisiología , Proteínas del Tejido Nervioso/metabolismo , Densidad Postsináptica , Proteoma/metabolismo , Animales , Hipocampo/fisiología , Hipocampo/ultraestructura , Ratones , Ratones Endogámicos DBA , Proteoma/fisiología , Proteoma/ultraestructura , Proteómica , Especificidad de la EspecieRESUMEN
The relation between protein structural alterations and tissue dysfunction is a major concern as protein fibrillation and/or aggregation due to structural alterations has been reported in many disease states. In the current study, Fourier transform infrared microspectroscopic imaging has been used to investigate diabetes-induced changes on protein secondary structure and macromolecular content in streptozotocin-induced diabetic rat liver. Protein secondary structural alterations were predicted using neural network approach utilizing the amide I region. Moreover, the role of selenium in the recovery of diabetes-induced alterations on macromolecular content and protein secondary structure was also studied. The results revealed that diabetes induced a decrease in lipid to protein and glycogen to protein ratios in diabetic livers. Significant alterations in protein secondary structure were observed with a decrease in α-helical and an increase in ß-sheet content. Both doses of selenium restored diabetes-induced changes in lipid to protein and glycogen to protein ratios. However, low-dose selenium supplementation was not sufficient to recover the effects of diabetes on protein secondary structure, while a higher dose of selenium fully restored diabetes-induced alterations in protein structure.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Hígado/metabolismo , Proteoma/química , Proteoma/ultraestructura , Selenio/administración & dosificación , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Animales , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/tratamiento farmacológico , Hígado/efectos de los fármacos , Masculino , Redes Neurales de la Computación , Proteoma/metabolismo , Ratas , Ratas Wistar , Recuperación de la Función/efectos de los fármacos , EstreptozocinaRESUMEN
To function properly protein molecules require both flexibility and rigidity, therefore fast and accurate prediction of protein rigidity/flexibility is one of the important problems in protein science. In this work we used two theoretical approaches to determine flexible regions in four homologous pairs of proteins from thermophilic and mesophilic organisms. Protein pairs chosen in this study were selected to represent four typical folding classes. Our first approach, FoldUnfold, uses amino acid sequence and statistical information on the density of contacts of amino acids in tertiary structures of known globular proteins. The main advantages of such knowledge-based methodology are its computational speed and ability to make predictions in the absence of three-dimensional (3D) structure of a protein. The second approach uses a graph theory-based rigid cluster decomposition termed FIRST, applied together with Molecular Dynamics (MD) simulations of proteins with known structure. While MD simulations are time-consuming, they are the most direct way of studying physical properties of proteins, including their rigidity/flexibility. Flexible regions predicted by both methods in this work were in good agreement with each other. We also showed that high mobility of a site is not necessarily indicative of its high flexibility and vice versa. In our simulations thermophile proteins were less flexible than their mesophilic homologues. Longer flexible loops were found in mesophilic proteins of all classes.
Asunto(s)
Algoritmos , Modelos Químicos , Modelos Moleculares , Proteoma/química , Proteoma/ultraestructura , Análisis de Secuencia de Proteína/métodos , Secuencia de Aminoácidos , Simulación por Computador , Datos de Secuencia Molecular , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Relación Estructura-ActividadRESUMEN
In this work we have studied the folding pathways for four pairs of homologous proteins from thermophilic and mesophilic organisms from two different structural classes (class a, all-alpha proteins and class d, alpha + beta proteins) using Monte Carlo simulations. We have obtained 50 trajectories for each protein and followed the free-energy profile and the order of folding of secondary structure elements between the last occurrence of the completely unfolded state and the first occurrence of the completely folded state. It turns out that the period of successful crossing of the free-energy barrier between unfolded and folded states for 40-45 trajectories (80-90%) makes 10% of the total folding time for four proteins (1tzvA, 1eyvA, 351c, and 1t4aA) and only 0.1% for two proteins (1dd3, 1ctf). This observation can be explained by a higher free-energy barrier for these proteins (1dd3, 1ctf) in comparison with other studied proteins. We have observed that folding pathways of thermophilic and mesophilic proteins may be the same, partly the same, and different. And similarity or difference between the folding pathways of thermophilic and mesophilic proteins does not depend on the structural class to which these proteins belong. Folding pathways for proteins from both classes correlate with the calculated folding nuclei for these proteins.
Asunto(s)
Algoritmos , Modelos Químicos , Modelos Moleculares , Proteoma/química , Proteoma/ultraestructura , Análisis de Secuencia de Proteína/métodos , Secuencia de Aminoácidos , Simulación por Computador , Modelos Estadísticos , Datos de Secuencia Molecular , Método de Montecarlo , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Relación Estructura-ActividadRESUMEN
Although silk is used to produce textiles and serves as a valuable biomaterial in medicine, information on silk proteins of the cocoon is limited. Scanning electron microscopy was applied to morphologically characterise the sample and the solubility of cocoon in lithium thiocyanate and 2-DE was carried out with multi-enzyme in-gel digestion followed by MS identification of silk-peptides. High-sequence coverage of the silk cocoon proteins fibroin light and heavy chain, sericins and fibrohexamerins was revealed and PTMs as heavy phosphorylation of silk fibroin heavy chain; lysine hydroxylation and Lys->allysine formation have been observed providing evidence for lysine-mediated cross linking of silk as found in collagens, which has not been reported so far. Tyrosine oxidation verified the presence of di-tyrosine cross links. A high degree of sequence conflicts probably representing single-nucleotide polymorphisms was observed. PTM and sequence conflicts may be modulating structure and physicochemical properties of silk.
Asunto(s)
Bombyx/química , Bombyx/metabolismo , Proteoma , Seda/química , Seda/metabolismo , Animales , Lisina/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Proteoma/química , Proteoma/metabolismo , Proteoma/ultraestructura , Seda/ultraestructuraRESUMEN
A relação entre estrutura e função protéica é um dos conceitos mais bem estabelecidos da biologia molecular. O acúmulo de evidências experimentais, cujos primeiros trabalhos datam de 1890, suportam essa hipótese com grande embasamento científico. Apesar da existência de evidências de mais de um século de estudos, somente no inicio da década de 90 começaram a surgir trabalhos mostrando de forma conclusiva a existência de proteínas funcionalmente ativas, mas incapazes de manter uma conformação estável em condições fisiológicas. Tais proteínas, hoje conhecidas como IUPs (do inglês Intrinsically Unstructured Proteins) estão envolvidas em importantes processos de saúde e doenças, tais como o câncer e diversos processos de interação parasito/hospedeiro. A presente dissertação tem como proposta o estabelecimento de um pipeline computacional visando à avaliação dos diferentes algoritmos de predição de desordem estrutural, seu desempenho e a posterior aplicação dessa ferramenta no estudo in silico do conteúdo de IUPs presentes no proteoma predito de Schistosoma mansoni. Complementarmente, foi desenhado um banco de dados MySQL capaz de albergar toda a informação de desordem estrutural juntamente com diferentes dados de caracterização das IUPs para S. mansoni.
Foram analisados um total de 10.417 proteínas, 7.373 predições de desordem estrutural, mais de 24.600 predições de características estruturais e funcionais, desenvolvidos 21 scripts, e todas essas predições e scripts desenvolvidos foram integrados em um pipeline totalmente automático e inédito para. análise de desordem estrutural. Nossas análises de sensibilidade e especificidade implementadas pela análise de gráficos ROC e pela integração de resultados utilizando bancos de dados relacionais indicam que a predição integrativa (consenso de quatro diferentes metodologias de predição) de desordem estrutural apresenta um ganho de 40% na correta identificação de regiões desordenadas se comparada às predições de cada metodologia individualmente. Aproximadamente 5,5% das regiões desordenadas identificadas tiveram suas coordenadas limítrofes ajustadas após comparação com as coordenadas de domínios conservados. Nossos resultados indicam que aproximadamente 33,6% do proteoma predito de S. mansoni apresenta desordem estrutural. Destas, 2% apresentam domínios transmembrana e 7% apresentam peptídeo sinal. A comparação do perfil funcional das IUPs com as proteínas globulares de S. mansoni demonstra uma maior proporção de IUPs envolvidas em processos de regulação celular e componentes extracelulares.
Asunto(s)
Biología Computacional/métodos , Esquistosomiasis mansoni/genética , Proteoma/ultraestructura , Schistosoma mansoni/ultraestructuraRESUMEN
A relação entre estrutura e função protéica é um dos conceitos mais bem estabelecidos da biologia molecular. O acúmulo de evidências experimentais, cujos primeiros trabalhos datam de 1890, suportam essa hipótese com grande embasamento científico. Apesar da existência de evidências de mais de um século de estudos, somente no inicio da década de 90 começaram a surgir trabalhos mostrando de forma conclusiva a existência de proteínas funcionalmente ativas, mas incapazes de manter uma conformação estável em condições fisiológicas. Tais proteínas, hoje conhecidas como IUPs (do inglês Intrinsically Unstructured Proteins) estão envolvidas em importantes processos de saúde e doenças, tais como o câncer e diversos processos de interação parasito/hospedeiro. A presente dissertação tem como proposta o estabelecimento de um pipeline computacional visando à avaliação dos diferentes algoritmos de predição de desordem estrutural, seu desempenho e a posterior aplicação dessa ferramenta no estudo in silico do conteúdo de IUPs presentes no proteoma predito de Schistosoma mansoni. Complementarmente, foi desenhado um banco de dados MySQL capaz de albergar toda a informação de desordem estrutural juntamente com diferentes dados de caracterização das IUPs para S. mansoni.
Foram analisados um total de 10.417 proteínas, 7.373 predições de desordem estrutural, mais de 24.600 predições de características estruturais e funcionais, desenvolvidos 21 scripts, e todas essas predições e scripts desenvolvidos foram integrados em um pipeline totalmente automático e inédito para. análise de desordem estrutural. Nossas análises de sensibilidade e especificidade implementadas pela análise de gráficos ROC e pela integração de resultados utilizando bancos de dados relacionais indicam que a predição integrativa (consenso de quatro diferentes metodologias de predição) de desordem estrutural apresenta um ganho de 40% na correta identificação de regiões desordenadas se comparada às predições de cada metodologia individualmente. Aproximadamente 5,5% das regiões desordenadas identificadas tiveram suas coordenadas limítrofes ajustadas após comparação com as coordenadas de domínios conservados. Nossos resultados indicam que aproximadamente 33,6% do proteoma predito de S. mansoni apresenta desordem estrutural. Destas, 2% apresentam domínios transmembrana e 7% apresentam peptídeo sinal. A comparação do perfil funcional das IUPs com as proteínas globulares de S. mansoni demonstra uma maior proporção de IUPs envolvidas em processos de regulação celular e componentes extracelulares.
Asunto(s)
Biología Computacional/métodos , Proteoma/ultraestructura , Schistosoma mansoni/ultraestructura , Esquistosomiasis mansoni/genéticaRESUMEN
MOTIVATION: The underlying assumption of many sequence-based comparative studies in proteomics is that different aspects of protein structure and therefore functionality may be linked to particular sequence motifs. This holds true if sequence similarity is sufficiently high, but in general the relationship between protein sequence and structure appears complex and is not well understood. RESULTS: Statistical analysis of multiple and pairwise structural alignments of protein structures within SCOP folds is performed. The results indicate that multiple conservation of residue identity is not common and that relationship between sequence and structure may be explained by a model based on the assumption that protein structure is tolerant to residue substitutions preserving hydropathic profile of the sequence. This model also explains the origin and specific value of the sequence similarity threshold, noticed in many previous studies, below which structural resemblance is not statistically expected.
Asunto(s)
Modelos Químicos , Modelos Moleculares , Proteoma/metabolismo , Proteoma/ultraestructura , Alineación de Secuencia/métodos , Análisis de Secuencia de Proteína/métodos , Secuencia de Aminoácidos , Simulación por Computador , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Estadística como Asunto , Relación Estructura-ActividadAsunto(s)
Humanos , Masculino , Femenino , Proteoma/análisis , Proteoma/aislamiento & purificación , Proteoma/ultraestructuraAsunto(s)
Masculino , Femenino , Humanos , Proteoma/análisis , Proteoma/aislamiento & purificación , Proteoma/ultraestructuraRESUMEN
Ulcerative colitis (UC) is a chronic inflammatory disorder primarily affecting the colon mucosa. Its etiology and pathogenesis remain unclear. We used 2-DE and MS to identify differentially expressed proteins among the UC active, UC inactive, nonspecific colitis, and normal colon mucosa. Thirteen down-regulated and six up-regulated proteins were identified. Of the down-expressed proteins, eight (heat-shock protein 90 (HSPA9B), heat-shock protein 60 (HSPD1), H+-transporting two-sector ATPase (ATP5B), prohibitin (PHB), mitochondrial malate dehydrogenase (MDH2), voltage-dependent anion-selective channel protein 1 (VDAC1), thioredoxin peroxidase (PRDX1), and thiol-specific antioxidant (PRDX2)) were mitochondrial proteins, three (ATP5B, MDH2, triosephosphate isomerase) were involved in energy generation, three (PRDX1, PRDX2, SELENBP1) were cellular antioxidants, and six (HSPD1, HSPA9B, PRDX1, PRDX2, PHB, VDAC1) were stress-response proteins. Transmission electron microscopy revealed pathological alterations of mitochondrial ultrastructures even before the global colonocyte changes in the UC colon mucosa. PHB, an essential mitochondrial component protein, was down-expressed in the disease active as well as inactive colon mucosa from the patients of UC, indicative of an early event of mitochondrial changes during UC development. In contrast, aberrant activation of NFAT and ectopic expression of potential immunogenic proteins (tumor rejection antigen 1 and poliovirus receptor related protein 1) were found in the UC-diseased colon mucosa. Our findings suggest the implications of colonocyte mitochondrial dysfunction and perturbed mucosa immune regulation in the pathogenesis of UC and provide potential targets for the development of a new therapy.
Asunto(s)
Colitis Ulcerosa/etiología , Colitis Ulcerosa/metabolismo , Proteoma/metabolismo , Proteómica , Adulto , Enfermedad Crónica , Colitis Ulcerosa/patología , Electroforesis en Gel Bidimensional , Humanos , Masculino , Prohibitinas , Proteoma/ultraestructuraRESUMEN
Organelle proteome has become one of the most important fields of proteomics, and the subcellular fractionation with high purity and yield has always been a challenge for cell biologists and also for the Human Liver Proteome Project (HLPP). The liver of a C57BL/6J mouse was chosen as the model to find the optimum method for subcellular preparation. The method we selected could obtain the multiple fractions including plasma membrane, mitochondria, nucleus, ER, and cytosol from a single homogenate. With the same procedure, it is for the first time that the preparation method of frozen homogenized livers was compared with that of the fresh livers and frozen livers. We systematically evaluated the purity, efficiency, and integrity by protein yield, immunoblotting, and transmission electron microscopy. Taken together, the method of multiple fractions from a single tissue is effective enough for subcellular fractionation of mouse liver. We give a selective sample preparation method for frozen homogenized livers, for rare clinical samples, which cannot easily be used for subcellular separation immediately. But the frozen livers are not recommended for organelles isolation. This result is especially useful for sample preparation of human liver for subcellular fractionation of HLPP.
Asunto(s)
Hígado/química , Hígado/metabolismo , Orgánulos/química , Proteoma/análisis , Proteómica/métodos , Animales , Humanos , Hígado/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Orgánulos/metabolismo , Orgánulos/ultraestructura , Proteoma/química , Proteoma/ultraestructura , Fracciones Subcelulares/química , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/ultraestructuraRESUMEN
Advances in gel-based nonradioactive protein expression and PTM detection using fluorophores has served as the impetus for developing analytical instrumentation with improved imaging capabilities. We describe a CCD camera-based imaging instrument, equipped with both a high-pressure Xenon arc lamp and a UV transilluminator, which provides broad-band wavelength coverage (380-700 nm and UV). With six-position filter wheels, both excitation and emission wavelengths may be selected, providing optimal measurement and quantitation of virtually any dye and allowing excellent spectral resolution among different fluorophores. While spatial resolution of conventional fixed CCD camera imaging systems is typically inferior to laser scanners, this problem is circumvented with the new instrument by mechanically scanning the CCD camera over the sample and collecting multiple images that are subsequently automatically reconstructed into a complete high-resolution image. By acquiring images in succession, as many as four different fluorophores may be evaluated from a gel. The imaging platform is suitable for analysis of the wide range of dyes and tags commonly encountered in proteomics investigations. The instrument is unique in its capabilities of scanning large areas at high resolution and providing accurate selectable illumination over the UV/visible spectral range, thus maximizing the efficiency of dye multiplexing protocols.
Asunto(s)
Fosfoproteínas/análisis , Proteínas/análisis , Proteoma/análisis , Proteómica/métodos , Proteínas de Arabidopsis/análisis , Colorantes , Cámaras gamma , Geles , Procesamiento de Imagen Asistido por Computador , Fosfoproteínas/ultraestructura , Proteínas/ultraestructura , Proteoma/ultraestructura , Sensibilidad y Especificidad , XenónRESUMEN
Imaging the proteome is a term that is used in many different contexts. The term implies that the entire cohort of proteins and their modifications are visualized. This unfortunately is not the case. In this mini-review, a concise overview is provided on different imaging technologies that are currently used to investigate the structure, function and dynamics of proteins and their organization. These techniques have been selected for review based on the unique insights they provide in subsets of the proteome. These techniques have been illustrated with practical examples of their merits. Mass spectrometry-based imaging technologies are playing a key role in proteome research and have been reviewed in more detail. They hold the promise of detailed molecular insight in the spatial organization of living system.
Asunto(s)
Proteoma/química , Proteoma/ultraestructura , Electroforesis en Gel Bidimensional , Procesamiento de Imagen Asistido por Computador , Microscopía de Contraste de Fase , Proteínas/química , Proteínas/aislamiento & purificación , Proteínas/ultraestructura , Proteoma/aislamiento & purificación , Proteómica , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
BACKGROUND: There is a need for a technology that can quantitatively assay multiple proteins from a single hair follicle while preserving the morphology of the follicle. For proteomic profiling, the technology should be less labor intensive, with a higher throughput, more quantitative and more reproducible than immunohistochemistry. OBJECTIVE: To test the ability of a novel method, layered expression scanning of hair (LES-hair) to detect the levels and localization of proteins in plucked hair follicles. METHODS: LES-hair was used to assay proteins in the plucked hair follicle. RESULTS: LES-hair detected differential expression of proteins within discrete regions of the plucked hair follicle. These proteins included cleaved caspase 3, Ki-67 and the phosphorylated forms of c-Kit, epidermal growth factor receptor and vascular endothelial growth factor receptor. CONCLUSION: LES-hair provides a research tool for studying the basic biology of plucked hair follicles and has potential clinical applications such as monitoring treatment of alopecia or using plucked hair follicles as a surrogate tissue to monitor pharmacodynamic effects of targeted cancer therapies.
Asunto(s)
Receptores ErbB/ultraestructura , Folículo Piloso/ultraestructura , Proteoma/ultraestructura , Adulto , Western Blotting , Técnicas de Cultivo , Receptores ErbB/metabolismo , Regulación de la Expresión Génica , Enfermedades del Cabello/diagnóstico , Enfermedades del Cabello/genética , Humanos , Masculino , Microscopía Electrónica de Rastreo , Proteínas/análisis , Proteínas/genética , Proteoma/genética , Muestreo , Sensibilidad y EspecificidadRESUMEN
Dendritic cells constitutively secrete a population of small (50-90 nm diameter) Ag-presenting vesicles called exosomes. When sensitized with tumor antigenic peptides, dendritic cells produce exosomes, which stimulate anti-tumor immune responses and the rejection of established tumors in mice. Using a systematic proteomic approach, we establish the first extensive protein map of a particular exosome population; 21 new exosomal proteins were thus identified. Most proteins present in exosomes are related to endocytic compartments. New exosomal residents include cytosolic proteins most likely involved in exosome biogenesis and function, mainly cytoskeleton-related (cofilin, profilin I, and elongation factor 1alpha) and intracellular membrane transport and signaling factors (such as several annexins, rab 7 and 11, rap1B, and syntenin). Importantly, we also identified a novel category of exosomal proteins related to apoptosis: thioredoxin peroxidase II, Alix, 14-3-3, and galectin-3. These findings led us to analyze possible structural relationships between exosomes and microvesicles released by apoptotic cells. We show that although they both represent secreted populations of membrane vesicles relevant to immune responses, exosomes and apoptotic vesicles are biochemically and morphologically distinct. Therefore, in addition to cytokines, dendritic cells produce a specific population of membrane vesicles, exosomes, with unique molecular composition and strong immunostimulating properties.