RESUMEN
RNA sequencing is a powerful method to build reference transcriptome assemblies and eventually sample-specific protein databases for mass-spectrometry-based analyses. This novel proteomics informed by transcriptomics (PIT) workflow improves proteome characterization of dynamic and especially nonmodel organism proteomes and moreover helps to identify novel gene products. With increasing popularity of such proteogenomics applications a growing number of researchers demand qualitative but resource-friendly and easy to use analysis strategies. Most PIT applications so far rely on the initially introduced Trinity de novo assembly tool. To aid potential users to start off with PIT, we compared main performance criteria of Trinity and other alternative RNA assembly tools known from the transcriptomics field including Oases, SOAPdenovo-Trans, and Trans-ABySS. Using exemplary data sets and software-specific default parameters, Trinity and alternative assemblers produced comparable and high-quality reference data for vertebrate transcriptomes/proteomes of varying complexity. However, Trinity required large computational resources and time. We found that alternative de novo assemblers, in particular, SOAPdenovo-Trans but also Oases and Trans-ABySS, rapidly produced protein databases with far lower computational requirements. By making choice among various RNA assembly tools, proteomics researchers new to transcriptome assembly and with future projects with high sample numbers can benefit from alternative approaches to efficiently apply PIT.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Proteómica/métodos , Análisis de Secuencia de ARN/métodos , Bases de Datos de Proteínas , Espectrometría de Masas , Programas InformáticosRESUMEN
Mass spectrometry-based methods allow for the direct, comprehensive analysis of expressed proteins and their quantification among different conditions. However, in general identification of proteins by assigning experimental mass spectra to peptide sequences of proteins relies on matching mass spectra to theoretical spectra derived from genomic databases of organisms. This conventional approach limits the applicability of proteomic methodologies to species for which a genome reference sequence is available. Recently, RNA-sequencing (RNA-Seq) became a valuable tool to overcome this limitation by de novo construction of databases for organisms for which no DNA sequence is available, or by refining existing genomic databases with transcriptomic data. Here we present a generic pipeline to make use of transcriptomic data for proteomics experiments. We show in particular how to efficiently fuel proteomic analysis workflows with sample-specific RNA-sequencing databases. This approach is useful for the proteomic analysis of so far unsequenced organisms, complex microbial metatranscriptomes/metaproteomes (for example in the human body), and for refining current proteomics data analysis that solely relies on the genomic sequence and predicted gene expression but not on validated gene products. Finally, the approach used in the here presented protocol can help to improve the data quality of conventional proteomics experiments that can be influenced by genetic variation or splicing events.
Asunto(s)
Biología Computacional/métodos , Bases de Datos Genéticas , Espectrometría de Masas , Proteoma , Proteómica/métodos , Análisis de Secuencia de ARN , Humanos , Anotación de Secuencia Molecular , Espectrometría de Masas en TándemRESUMEN
Nutrition is a basic component of life. Nowadays, human nutrition research focuses amongst others on health-related aspects of food ingredients and extracts, and on analyzing the outcomes of specific diets. Usually, food ingredients such as bioactive peptides come in complex matrices. Single compounds, multiple interactions thereof and the underlying food matrix can vary physiological response of the organism. Proteins and peptides derived from food and beverages can cause adverse allergic reactions but are in general required for multiple functions such as growth and homeostatic regulation. Endogenously expressed human proteins and peptides can be used as biomarkers to monitor physiological deregulation and the effects of food consumption. The intestinal microbiome seems to play a fundamental role in establishing and maintaining physiological regulation and in digesting proteins and peptides and other biomolecules derived from food. Notably, the subtle interplay of flavor naturals in food and beverages with olfactory receptors can result in establishing human taste preferences, which again influences overall physiology. This article presents basic approaches and concepts to address scientific questions in nutritional proteomics and discusses potential benefits of proteomics-based methodologies to help advance the field of molecular nutrition research.
Asunto(s)
Biología Computacional , Ciencias de la Nutrición , Espectrometría de MasasRESUMEN
Phytoplasmas are pathogenic bacteria within the class of Mollicutes, which are associated with more than 1000 plant diseases. In this study, we applied quantitative mass spectrometry to analyse affected pathways of the model plant tobacco (Nicotiana occidentalis) upon Candidatus Phytoplasma mali strain AT infection. Using tissue obtained from leaf midribs, 1466 plant-assigned proteins were identified. For 1019 of these proteins, we could reproducibly quantify the expression changes of infected versus noninfected plants, of which 157 proteins were up- and 173 proteins were downregulated. Differential expression took place in a number of pathways, among others strong downregulation of porphyrin and chlorophyll metabolism and upregulation of alpha-linolenic acid metabolism, which was consistent with observed increased levels of jasmonic acid, a key signal molecule of plant defence. Our data shed light on the molecular networks that are involved in defence of plants against phytoplasma infection and provide a resource for further studies.
Asunto(s)
Interacciones Huésped-Patógeno , Nicotiana/metabolismo , Nicotiana/microbiología , Phytoplasma/fisiología , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/metabolismo , Proteómica/métodos , Ciclopentanos/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica de las Plantas , Oxilipinas/metabolismo , Enfermedades de las Plantas/genética , Proteínas de Plantas/genética , Transducción de Señal , Nicotiana/genéticaRESUMEN
'Candidatus Phytoplasma mali' is a phytopathogenic bacterium of the family Acholeplasmataceae assigned to the class Mollicutes. This causative agent of the apple proliferation colonizes in Malus domestica the sieve tubes of the plant phloem resulting in a range of symptoms such as witches'--broom formation, reduced vigor and affecting size and quality of the crop. The disease is responsible for strong economical losses in Europe. Although the genome sequence of the pathogen is available, there is only limited information on expression of selected genes and metabolic key features that have not been examined on the transcriptomic or proteomic level so far. This situation is similar to many other phytoplasmas. In the work presented here, RNA-Seq and mass spectrometry shotgun techniques were applied on tissue samples from Nicotiana occidentalis infected by 'Ca. P. mali' strain AT providing insights into transcriptome and proteome of the pathogen. Data analysis highlights expression of 208 genes including 14 proteins located in the terminal inverted repeats of the linear chromosome. Beside a high portion of house keeping genes, the recently discussed chaperone GroES/GroEL is expressed. Furthermore, gene expression involved in formation of a type IVB and of the Sec-dependent secretion system was identified as well as the highly expressed putative pathogenicity-related SAP11-like effector protein. Metabolism of phytoplasmas depends on the uptake of spermidine/putescine, amino acids, co-factors, carbohydrates and in particular malate/citrate. The expression of these transporters was confirmed and the analysis of the carbohydrate cycle supports the suggested alternative energy-providing pathway for phytoplasmas releasing acetate and providing ATP. The phylogenetic analyses of malate dehydrogenase and acetate kinase in phytoplasmas show a closer relatedness to the Firmicutes in comparison to Mycoplasma species indicating an early divergence of the Acholeplasmataceae from the Mollicutes.