Proteome data of Anopheles stephensi hemolymph using high resolution mass spectrometry.
ABSTRACT: The article provides insights into the protein expression in Anopheles stephensi hemolymph. We carried out data acquisition using a high-resolution LTQ-Orbitrap Velos mass spectrometer to identify the hemolymph proteins of An. stephensi. Experimentally derived mass spectrometry data was analyzed using Proteome Discoverer 2.1 software using two different search algorithms SEQUEST and MASCOT. A total of 1091 proteins were identified from the hemolymph. The identified proteins were categorized for their role in biological processes and molecular functions. The interactions between these proteins were predicted using STRING online tool. Relation can be drawn between the data provided in this study to the already published article "Integrating transcriptomics and proteomics data for accurate assembly and annotation of genomes" (Prasad et al., 2017) .
Project description:The data article reports data of the proteins expressed in female Anopheles stephensi salivary glands. Proteomic data were acquired using high-resolution mass spectrometers - Orbitrap-Velos and Orbitrap-Elite. Samples derived from adult female A. stephensi salivary glands led to the identification of 4390 proteins. Mass spectrometry data were analyzed on Proteome Discoverer (Version 2.1) platform with Sequest and Mascot search engines. The identified proteins were analyzed for their Gene Ontology annotation, interaction network and their possible roles in vector-parasite interaction. The data provided here are related to our published article "Integrating transcriptomics and proteomics data for accurate assembly and annotation of genomes" (Prasad et al., 2017) .
Project description:This article contains data on the proteins expressed in the ovaries of Anopheles stephensi, a major vector of malaria in India. Data acquisition was performed using a high-resolution Orbitrap-Velos mass spectrometer. The acquired MS/MS data was searched against An. stephensi protein database comprising of 11,789 sequences. Overall, 4407 proteins were identified, functional analysis was performed for the identified proteins and a protein-protein interaction map predicted. The data provided here is also related to a published article - "Integrating transcriptomics and proteomics data for accurate assembly and annotation of genomes" (Prasad et al., 2017) .
Project description:Fat body from Anopheles stephensi female mosquitoes were dissected and processed for proteomic analysis. Both SDS-PAGE and basic Reverse Phase Liquid Chromatography-based fractionation strategies were used to achieve a broad coverage of protein identification. The fractionated peptides were then analyzed on a high-resolution mass spectrometer. Searching the raw data against the protein database of An. stephensi resulted in identification of 4535 proteins, which is, to our knowledge, the largest catalog of fat body proteome in any mosquito vector species reported so far. Bioinformatics analysis on these fat body proteins suggested the enrichment of biological processes including carbon and lipid metabolism, amino acid metabolism, signal peptide processing and oxidation-reduction. In addition, using proteogenomic approaches, 43 novel proteins were identified, which were not listed in the annotated gene annotations of An. stephensi. The data used in the analysis are related to the article 'Integrating transcriptomic and proteomic data for accurate assembly and annotation of genomes' (Prasad et al., 2017).
Project description:Anopheles stephensi Liston is one of the major vectors of malaria in urban areas of India. Midgut plays a central role in the vector life cycle and transmission of malaria. Because gene expression of An. stephensi midgut has not been investigated at protein level, an unbiased mass spectrometry-based proteomic analysis of midgut tissue was carried out. Midgut tissue proteins from female An. stephensi mosquitoes were extracted using 0.5% SDS and digested with trypsin using two complementary approaches, in-gel and in-solution digestion. Fractions were analysed on high-resolution mass spectrometer, which resulted in acquisition of 494,960 MS/MS spectra. The MS/MS spectra were searched against protein database comprising of known and predicted proteins reported in An. stephensi using Sequest and Mascot software. In all, 47,438 peptides were identified corresponding to 5,709 An. stephensi proteins. The identified proteins were functionally categorized based on their cellular localization, biological processes and molecular functions using Gene Ontology (GO) annotation. Several proteins identified in this data are known to mediate the interaction of the Plasmodium with vector midgut and also regulate parasite maturation inside the vector host. This study provides information about the protein composition in midgut tissue of female An. stephensi, which would be useful in understanding vector parasite interaction at molecular level and besides being useful in devising malaria transmission blocking strategies. The data of this study is related to the research article "Integrating transcriptomics and proteomics data for accurate assembly and annotation of genomes".
Project description:Antennae of female Anopheles stephensi mosquitoes were dissected and lysed with 1% SDS. Proteins were extracted using ultra sonication and analyzed on high resolution mass spectrometer. Proteomic data was analyzed using two search algorithms SEQUEST and Mascot, resulting in the identification of 22,729 peptides corresponding to 3262 proteins. These proteins were characterized using different bioinformatics tools. VectorBase resource was used to assign Gene Ontology (GO) terms. Using Biomart tool ortholog information was fetched from the VectorBase database. Raw mass spectrometric data was deposited in ProteomeXchange Consortium via PRIDE partner repository in the public dataset PXD001128. Proteins involved in insecticide resistance and odorant binding were the most abundant in the antennae. The proteins identified in this study could be targeted for developing novel vector control strategy.
Project description:The two key steps for analyzing proteomic data generated by high-resolution MS are database searching and postprocessing. While the two steps are interrelated, studies on their combinatory effects and the optimization of these procedures have not been adequately conducted. Here, we investigated the performance of three popular search engines (SEQUEST, Mascot, and MS Amanda) in conjunction with five filtering approaches, including respective score-based filtering, a group-based approach, local false discovery rate (LFDR), PeptideProphet, and Percolator. A total of eight data sets from various proteomes (e.g., E. coli, yeast, and human) produced by various instruments with high-accuracy survey scan (MS1) and high- or low-accuracy fragment ion scan (MS2) (LTQ-Orbitrap, Orbitrap-Velos, Orbitrap-Elite, Q-Exactive, Orbitrap-Fusion, and Q-TOF) were analyzed. It was found combinations involving Percolator achieved markedly more peptide and protein identifications at the same FDR level than the other 12 combinations for all data sets. Among these, combinations of SEQUEST-Percolator and MS Amanda-Percolator provided slightly better performances for data sets with low-accuracy MS2 (ion trap or IT) and high accuracy MS2 (Orbitrap or TOF), respectively, than did other methods. For approaches without Percolator, SEQUEST-group performs the best for data sets with MS2 produced by collision-induced dissociation (CID) and IT analysis; Mascot-LFDR gives more identifications for data sets generated by higher-energy collisional dissociation (HCD) and analyzed in Orbitrap (HCD-OT) and in Orbitrap Fusion (HCD-IT); MS Amanda-Group excels for the Q-TOF data set and the Orbitrap Velos HCD-OT data set. Therefore, if Percolator was not used, a specific combination should be applied for each type of data set. Moreover, a higher percentage of multiple-peptide proteins and lower variation of protein spectral counts were observed when analyzing technical replicates using Percolator-associated combinations; therefore, Percolator enhanced the reliability for both identification and quantification. The analyses were performed using the specific programs embedded in Proteome Discoverer, Scaffold, and an in-house algorithm (BuildSummary). These results provide valuable guidelines for the optimal interpretation of proteomic results and the development of fit-for-purpose protocols under different situations.
Project description:The transmission of drug-resistant parasites by the mosquito may be influenced by the altered biological fitness of drug-resistant parasites and different immune reactions or metabolic change in the mosquito. At this point, little is known about the variations in mosquito immunity and metabolism when mosquitoes are infected with drug-resistant parasites. To understand the differential gene expression in Anopheles following infection with drug-resistant Plasmodium, we conducted a genome-wide transcriptomic profiling analysis of Anopheles stephensi following feeding on mice with drug-resistant or drug-sensitive P. yoelii, observed changes in gene expression profiles and identified transcripts affected by the drug-resistant parasite.To study the impact of drug-resistant Plasmodium infections on An. stephensi gene transcription, we analyzed the three major transition stages of Plasmodium infections: at 24 h and 13 and 19 days after blood-feeding. Six cDNA libraries (R-As24h, R-As13d, R-As19d,S-As24h, S-As13dand S-As19d) were constructed, and RNA sequencing was subsequently performed. In total, approximately 50.1 million raw reads, 47.9 million clean reads and 7.18G clean bases were obtained. Following differentially expressed gene (DEG) analysis, GO enrichment analysis of DEGs, and functional classification by KEGG, we showed that the variations in gene expression in An. stephensi infected by the drug-resistant P. yoelii NSM occurred mainly at 13 days after blood meal during sporozoite migration through the hemolymph. The differentially expressed genes included those functioning in some important immune reaction and iron metabolism pathways, such as pattern recognition receptors, regulators of the JNK pathway, components of the phagosome pathway, regulators of the melanization response, activators of complement reactions, insulin signaling cascade members, oxidative stress and detoxification proteins.Our study shows that drug-resistant P. yoelii NSM has an impact on the transcript abundance levels of An.stephensi mostly at 13 days after blood meal during sporozoite migration through the hemolymph and that most differentially expressed genes were downregulated. Our results highlight the need for a better understanding of selective pressures from these differentially expressed genes of the drug-resistant Plasmodium in the mosquito and the different transmission patterns of drug-resistant Plasmodium by Anopheles.
Project description:Salivary gland proteins of Anopheles mosquitoes offer attractive targets to understand interactions with sporozoites, blood feeding behavior, homeostasis, and immunological evaluation of malaria vectors and parasite interactions. To date limited studies have been carried out to elucidate salivary proteins of An. stephensi salivary glands. The aim of the present study was to provide detailed analytical attributives of functional salivary gland proteins of urban malaria vector An. stephensi. A proteomic approach combining one-dimensional electrophoresis (1DE), ion trap liquid chromatography mass spectrometry (LC/MS/MS), and computational bioinformatic analysis was adopted to provide the first direct insight into identification and functional characterization of known salivary proteins and novel salivary proteins of An. stephensi. Computational studies by online servers, namely, MASCOT and OMSSA algorithms, identified a total of 36 known salivary proteins and 123 novel proteins analysed by LC/MS/MS. This first report describes a baseline proteomic catalogue of 159 salivary proteins belonging to various categories of signal transduction, regulation of blood coagulation cascade, and various immune and energy pathways of An. stephensi sialotranscriptome by mass spectrometry. Our results may serve as basis to provide a putative functional role of proteins in concept of blood feeding, biting behavior, and other aspects of vector-parasite host interactions for parasite development in anopheline mosquitoes.
Project description:The co-operation of specialized organ systems in complex multicellular organisms depends on effective chemical communication. Thus, body fluids (like blood, lymph or intraspinal fluid) contain myriads of signaling mediators apart from metabolites. Moreover, these fluids are also of crucial importance for immune and wound responses. Compositional analyses of human body fluids are therefore of paramount diagnostic importance. Further improving their comprehensiveness should increase our understanding of inter-organ communication. In arthropods, which have trachea for gas exchange and an open circulatory system, the single dominating interstitial fluid is the hemolymph. Accordingly, a detailed analysis of hemolymph composition should provide an especially comprehensive picture of chemical communication and defense in animals. Therefore we used an extensive protein fractionation workflow in combination with a discovery-driven proteomic approach to map out the detectable protein composition of hemolymph isolated from Drosophila larvae. Combined mass spectrometric analysis revealed more than 700 proteins extending far beyond the previously known Drosophila hemolymph proteome. Moreover, by comparing hemolymph isolated from either fed or starved larvae, we provide initial provisional insights concerning compositional changes in response to nutritional state. Storage proteins in particular were observed to be strongly reduced by starvation. Our hemolymph proteome catalog provides a rich basis for data mining, as exemplified by our identification of potential novel cytokines, as well as for future quantitative analyses by targeted proteomics.
Project description:Neuropeptides are often released into circulatory fluid (hemolymph) to act as circulating hormones and regulate many physiological processes. However, the detection of these low-level peptide hormones in circulation is often complicated by high salt interference and rapid degradation of proteins and peptides in crude hemolymph extracts. In this study, we systematically evaluated three different neuropeptide extraction protocols and developed a simple and effective hemolymph preparation method suitable for MALDI MS profiling of neuropeptides by combining acid-induced abundant protein precipitation/depletion, ultrafiltration, and C(18) micro-column desalting. In hemolymph samples collected from the crab Cancer borealis, several secreted neuropeptides have been detected, including members from at least five neuropeptide families, such as RFamide, allatostatin, orcokinin, tachykinin-related peptide (TRP), and crustacean cardioactive peptide (CCAP). Furthermore, two TRPs were detected in the hemolymph collected from food-deprived animals, suggesting the potential role of these neuropeptides in feeding regulation. In addition, a novel peptide with a Lys-Phe-amide C-terminus was identified and de novo sequenced directly from the Cancer borealis hemolymph sample. To better characterize the hemolymph peptidome, we also identified several abundant peptide signals in C. borealis hemolymph that were assigned to protein degradation products. Collectively, our study describes a simple and effective sample preparation method for neuropeptide analysis directly from crude crustacean hemolymph. Numerous endogenous neuropeptides were detected, including both known ones and new peptides whose functions remain to be characterized.