RESUMO
Natural genetic variation is the raw material of evolution and influences disease development and progression. An important question is how this genetic variation translates into variation in protein abundance. To analyze the effects of the genetic background on gene and protein expression in the nematode Caenorhabditis elegans, we quantitatively compared the two genetically highly divergent wild-type strains N2 and CB4856. Gene expression was analyzed by microarray assays, and proteins were quantified using stable isotope labeling by amino acids in cell culture. Among all transcribed genes, we found 1,532 genes to be differentially transcribed between the two wild types. Of the total 3,238 quantified proteins, 129 proteins were significantly differentially expressed between N2 and CB4856. The differentially expressed proteins were enriched for genes that function in insulin-signaling and stress-response pathways, underlining strong divergence of these pathways in nematodes. The protein abundance of the two wild-type strains correlates more strongly than protein abundance versus transcript abundance within each wild type. Our findings indicate that in C. elegans only a fraction of the changes in protein abundance can be explained by the changes in mRNA abundance. These findings corroborate with the observations made across species.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Perfilação da Expressão Gênica/métodos , Variação Genética , Proteômica/métodos , Animais , Evolução Biológica , Caenorhabditis elegans/classificação , Proteínas de Caenorhabditis elegans/genética , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Marcação por Isótopo/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodosRESUMO
Complex traits, including common disease-related traits, are affected by many different genes that function in multiple pathways and networks. The apoptosis, MAPK, Notch, and Wnt signalling pathways play important roles in development and disease progression. At the moment we have a poor understanding of how allelic variation affects gene expression in these pathways at the level of translation. Here we report the effect of natural genetic variation on transcript and protein abundance involved in developmental signalling pathways in Caenorhabditis elegans. We used selected reaction monitoring to analyse proteins from the abovementioned four pathways in a set of recombinant inbred lines (RILs) generated from the wild-type strains N2 (Bristol) and CB4856 (Hawaii) to enable quantitative trait locus (QTL) mapping. About half of the cases from the 44 genes tested showed a statistically significant change in protein abundance between various strains, most of these were however very weak (below 1.3-fold change). We detected a distant QTL on the left arm of chromosome II that affected protein abundance of the phosphatidylserine receptor protein PSR-1, and two separate QTLs that influenced embryonic and ionizing radiation-induced apoptosis on chromosome IV. Our results demonstrate that natural variation in C. elegans is sufficient to cause significant changes in signalling pathways both at the gene expression (transcript and protein abundance) and phenotypic levels.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Animais , Apoptose , Caenorhabditis elegans/citologia , Mapeamento Cromossômico , Regulação da Expressão Gênica no Desenvolvimento , Variação Genética , Locos de Características Quantitativas , Transdução de Sinais , Ativação TranscricionalRESUMO
Model organisms are an important tool for the development and validation of analytical approaches for proteomics and for the study of basic mechanisms of biological processes. The Initiative on Model Organism Proteomics (iMOP) organized a session during the 11th HUPO World Congress in Boston in 2012, highlighting the potential of proteomics studies in model organism for the elucidation of important mechanisms regulating the interaction of humans with its environment. Major subjects were the use of model organisms for the study of molecular events triggering the interaction of host organisms with the surrounding microbiota and the elucidation of the complex influence of nutrition on the health of human beings.
Assuntos
Modelos Animais , Modelos Biológicos , Proteômica/métodos , Animais , Bactérias/genética , Bactérias/imunologia , Humanos , Insetos/genética , Insetos/imunologia , Microbiota/genética , Fenômenos Fisiológicos da Nutrição , Plantas/genética , Proteoma/análise , Proteoma/genética , Proteoma/metabolismoRESUMO
iMOP--the Initiative on Model Organism Proteomes--was accepted as a new HUPO initiative at the Ninth HUPO meeting in Sydney in 2010. A goal of iMOP is to integrate research groups working on a great diversity of species into a model organism community. At the Tenth HUPO meeting in Geneva this variety was reflected in the iMOP session on Tuesday September 6, 2011. The presentations covered the quantitative proteome database PaxDb, proteomics projects studying farm animals, Arabidopsis thaliana, as well as host-pathogen interactions.
Assuntos
Bases de Dados como Assunto , Interações Hospedeiro-Patógeno , Proteoma , Animais , Animais Domésticos , Arabidopsis/química , SuíçaRESUMO
The community working on model organisms is growing steadily and the number of model organisms for which proteome data are being generated is continuously increasing. To standardize efforts and to make optimal use of proteomics data acquired from model organisms, a new Human Proteome Organisation (HUPO) initiative on model organism proteomes (iMOP) was approved at the HUPO Ninth Annual World Congress in Sydney, 2010. iMOP will seek to stimulate scientific exchange and disseminate HUPO best practices. The needs of model organism researchers for central databases will be better represented, catalyzing the integration of proteomics and organism-specific databases. Full details of iMOP activities, members, tools and resources can be found at our website http://www.imop.uzh.ch/ and new members are invited to join us.
Assuntos
Arabidopsis/química , Modelos Animais , Proteoma , Animais , Animais de Laboratório , Bases de Dados de Proteínas , HumanosRESUMO
Efficient experimental strategies are needed to validate computationally predicted microRNA (miRNA) target genes. Here we present a large-scale targeted proteomics approach to validate predicted miRNA targets in Caenorhabditis elegans. Using selected reaction monitoring (SRM), we quantified 161 proteins of interest in extracts from wild-type and let-7 mutant worms. We demonstrate by independent experimental downstream analyses such as genetic interaction, as well as polysomal profiling and luciferase assays, that validation by targeted proteomics substantially enriched for biologically relevant let-7 interactors. For example, we found that the zinc finger protein ZTF-7 was a bona fide let-7 miRNA target. We also validated predicted miR-58 targets, demonstrating that this approach is adaptable to other miRNAs. We propose that targeted mass spectrometry can be applied generally to validate candidate lists generated by computational methods or in large-scale experiments, and that the described strategy should be readily adaptable to other organisms.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , MicroRNAs/genética , Modelos Genéticos , Proteômica/métodos , Animais , Sequência de Bases , Caenorhabditis elegans/metabolismo , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Genes de Helmintos , Luciferases/genética , Espectrometria de Massas , MicroRNAs/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da EspécieAssuntos
Bactérias/metabolismo , Eucariotos/metabolismo , Modelos Biológicos , Proteoma/análise , Proteômica/métodos , Bactérias/genética , Proteínas de Bactérias/análise , Eletroforese em Gel Bidimensional/métodos , Eucariotos/genética , Proteínas Fúngicas/análise , Espectrometria de Massas/métodos , Modelos Animais , Processamento de Proteína Pós-TraducionalRESUMO
The nematode Caenorhabditis elegans (C. elegans) has been used with much success to study a number of biological processes. Although mostly known for its powerful forward and reverse genetics, work from many different groups over the past years has allowed this model organism to develop into a respectable system for proteomics studies as well. Large-scale survey studies led to improved genome annotation and to the generation of proteome catalogs, which set the stage for subsequent targeted proteomics studies. A number of focused comparative studies contributed to a better understanding of insulin signaling, spermatogenesis, oogenesis, and differential gene expression during development. In addition, C. elegans subproteomes and posttranslational modifications like glycosylation and phosphorylation have been identified. Here we describe the history of C. elegans proteomics, and provide a survey of the different methods that have been applied for relative and absolute quantification in comparative and global protein profiling studies in the worm. These studies suggest that C. elegans will provide a rich trove for "worm proteomicists".
Assuntos
Proteínas de Caenorhabditis elegans/análise , Caenorhabditis elegans/química , Proteômica/métodos , Animais , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Insulina/genética , Insulina/fisiologia , Oogênese/genética , Oogênese/fisiologia , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/fisiologia , Espermatogênese/genética , Espermatogênese/fisiologiaRESUMO
Comprehensive characterization of a proteome is a fundamental goal in proteomics. To achieve saturation coverage of a proteome or specific subproteome via tandem mass spectrometric identification of tryptic protein sample digests, proteomics data sets are growing dramatically in size and heterogeneity. The trend toward very large integrated data sets poses so far unsolved challenges to control the uncertainty of protein identifications going beyond well established confidence measures for peptide-spectrum matches. We present MAYU, a novel strategy that reliably estimates false discovery rates for protein identifications in large scale data sets. We validated and applied MAYU using various large proteomics data sets. The data show that the size of the data set has an important and previously underestimated impact on the reliability of protein identifications. We particularly found that protein false discovery rates are significantly elevated compared with those of peptide-spectrum matches. The function provided by MAYU is critical to control the quality of proteome data repositories and thereby to enhance any study relying on these data sources. The MAYU software is available as standalone software and also integrated into the Trans-Proteomic Pipeline.
Assuntos
Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Algoritmos , Animais , Caenorhabditis elegans/metabolismo , Biologia Computacional/métodos , Bases de Dados de Proteínas , Reações Falso-Positivas , Leptospira/metabolismo , Modelos Estatísticos , Peptídeos/química , Proteoma , Reprodutibilidade dos Testes , Schizosaccharomyces/metabolismoRESUMO
The nematode Caenorhabditis elegans is a popular model system in genetics, not least because a majority of human disease genes are conserved in C. elegans. To generate a comprehensive inventory of its expressed proteome, we performed extensive shotgun proteomics and identified more than half of all predicted C. elegans proteins. This allowed us to confirm and extend genome annotations, characterize the role of operons in C. elegans, and semiquantitatively infer abundance levels for thousands of proteins. Furthermore, for the first time to our knowledge, we were able to compare two animal proteomes (C. elegans and Drosophila melanogaster). We found that the abundances of orthologous proteins in metazoans correlate remarkably well, better than protein abundance versus transcript abundance within each organism or transcript abundances across organisms; this suggests that changes in transcript abundance may have been partially offset during evolution by opposing changes in protein abundance.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Proteoma , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Drosophila/genética , Duplicação Gênica , Genoma , Óperon , Proteômica/métodos , Homologia de Sequência de AminoácidosRESUMO
Synaptosomes are isolated synapses produced by subcellular fractionation of brain tissue. They contain the complete presynaptic terminal, including mitochondria and synaptic vesicles, and portions of the postsynaptic side, including the postsynaptic membrane and the postsynaptic density (PSyD). A proteomic characterisation of synaptosomes isolated from mouse brain was performed employing the isotope-coded affinity tag (ICAT) method and tandem mass spectrometry (MS/MS). After isotopic labelling and tryptic digestion, peptides were fractionated by cation exchange chromatography and cysteine-containing peptides were isolated by affinity chromatography. The peptides were identified by microcapillary liquid chromatography-electrospray ionisation MS/MS (muLC-ESI MS/MS). In two experiments, peptides representing a total of 1131 database entries were identified. They are involved in different presynaptic and postsynaptic functions, including synaptic vesicle exocytosis for neurotransmitter release, vesicle endocytosis for synaptic vesicle recycling, as well as postsynaptic receptors and proteins constituting the PSyD. Moreover, a large number of soluble and membrane-bound molecules serving functions in synaptic signal transduction and metabolism were detected. The results provide an inventory of the synaptic proteome and confirm the suitability of the ICAT method for the assessment of synaptic structure, function and plasticity.