Gourav Dey

A draft map of the human proteome

The availability of human genome sequence has transformed biomedical research over the past decade. However, an equivalent map for the human proteome with direct measurements of proteins and peptides does not exist yet. Here we present a draft map of the human proteome using high-resolution Fourier-transform mass spectrometry. In-depth proteomic profiling of 30 histologically normal human samples, including 17 adult tissues, 7 fetal tissues and 6 purified primary haematopoietic cells, resulted in identification of proteins encoded by 17,294 genes accounting for approximately 84% of the total annotated protein-coding genes in humans. A unique and comprehensive strategy for proteogenomic analysis enabled us to discover a number of novel protein-coding regions, which includes translated pseudogenes, non-coding RNAs and upstream open reading frames. This large human proteome catalogue (available as an interactive web-based resource at http://www.humanproteomemap.org) will complement available human genome and transcriptome data to accelerate biomedical research in health and disease.

Annotation of the Zebrafish Genome through an Integrated Transcriptomic and Proteomic Analysis

Accurate annotation of protein-coding genes is one of the primary tasks upon the completion of whole genome sequencing of any organism. In this study, we used an integrated transcriptomic and proteomic strategy to validate and improve the existing zebrafish genome annotation. We undertook high-resolution mass-spectrometry-based proteomic profiling of 10 adult organs, whole adult fish body, and two developmental stages of zebrafish (SAT line), in addition to transcriptomic profiling of six organs. More than 7,000 proteins were identified from proteomic analyses, and ∼69,000 high-confidence transcripts were assembled from the RNA sequencing data. Approximately 15% of the transcripts mapped to intergenic regions, the majority of which are likely long non-coding RNAs. These high-quality transcriptomic and proteomic data were used to manually reannotate the zebrafish genome. We report the identification of 157 novel protein-coding genes. In addition, our data led to modification of existing gene structures including novel exons, changes in exon coordinates, changes in frame of translation, translation in annotated UTRs, and joining of genes. Finally, we discovered four instances of genome assembly errors that were supported by both proteomic and transcriptomic data. Our study shows how an integrative analysis of the transcriptome and the proteome can extend our understanding of even well-annotated genomes.

A compendium of molecules involved in vector-pathogen interactions pertaining to malaria

Malaria is a vector-borne disease causing extensive morbidity, debility and mortality. Development of resistance to drugs among parasites and to conventional insecticides among vector-mosquitoes necessitates innovative measures to combat this disease. Identification of molecules involved in the maintenance of complex developmental cycles of the parasites within the vector and the host can provide attractive targets to intervene in the disease transmission. In the last decade, several efforts have been made in identifying such molecules involved in mosquito-parasite interactions and, subsequently, validating their role in the development of parasites within the vector. In this study, a list of mosquito proteins, which facilitate or inhibit the development of malaria parasites in the midgut, haemolymph and salivary glands of mosquitoes, is compiled. A total of 94 molecules have been reported and validated for their role in the development of malaria parasites inside the vector. This compendium of molecules will serve as a centralized resource to biomedical researchers investigating vector-pathogen interactions and malaria transmission.

Characterizing the normal proteome of human ciliary body

BackgroundThe ciliary body is the circumferential muscular tissue located just behind the iris in the anterior chamber of the eye. It plays a pivotal role in the production of aqueous humor, maintenance of the lens zonules and accommodation by changing the shape of the crystalline lens. The ciliary body is the major target of drugs against glaucoma as its inhibition leads to a drop in intraocular pressure. A molecular study of the ciliary body could provide a better understanding about the pathophysiological processes that occur in glaucoma. Thus far, no large-scale proteomic investigation has been reported for the human ciliary body.ResultsIn this study, we have carried out an in-depth LC-MS/MS-based proteomic analysis of normal human ciliary body and have identified 2,815 proteins. We identified a number of proteins that were previously not described in the ciliary body including importin 5 (IPO5), atlastin-2 (ATL2), B-cell receptor associated protein 29 (BCAP29), basigin (BSG), calpain-1 (CAPN1), copine 6 (CPNE6), fibulin 1 (FBLN1) and galectin 1 (LGALS1). We compared the plasma proteome with the ciliary body proteome and found that the large majority of proteins in the ciliary body were also detectable in the plasma while 896 proteins were unique to the ciliary body. We also classified proteins using pathway enrichment analysis and found most of proteins associated with ubiquitin pathway, EIF2 signaling, glycolysis and gluconeogenesis.ConclusionsMore than 95% of the identified proteins have not been previously described in the ciliary body proteome. This is the largest catalogue of proteins reported thus far in the ciliary body that should provide new insights into our understanding of the factors involved in maintaining the secretion of aqueous humor. The identification of these proteins will aid in understanding various eye diseases of the anterior segment such as glaucoma and presbyopia.

Signaling network of Oncostatin M pathway

Oncostatin M (OSM), belonging to the IL-6 family of cytokines (Heinrich et al. 2003), was first reported and purified from U937 monocytic cells (Zarling et al. 1986; Ensoli et al. 1999; Hasegawa et al. 1999). In normal physiological condition, OSM is associated with multiple biological processes and cellular responses including growth, differentiation, and inflammation However, anti-proliferative activity of OSM against breast cancer cell line generated the interest of biomedical community on this molecule (Douglas et al. 1997, 1998). OSM was also found associated with pathological conditions such as proliferation of ovarian cancer cells (Taga and Kishimoto 1997), prostate cancer 22Rv1 cells (Hoffman et al. 1996), up-regulation of the ER chaperone Grp78/BiP in the liver cells, atherosclerotic lesions, ischemic heart disease and rheumatoid arthritis (Linsley et al. 1990; Dunham et al. 1999). The dual role of OSM in either inducing or inhibiting the proliferation of various types of cells called upon the scientific community to investigate role of OSM in various physiological and experimental contexts in detail. However, diverse molecular level information pertaining to OSM signaling is not available in a centralized resource. Therefore, we have systematically gathered and curated molecular information from literature and created a public resource for OSM induced signaling events. We integrated OSM signaling pathway into NetPath (Kandasamy et al. 2010), which is a public resource of human signaling pathways. OSM is known to mediate its biological effects by binding to two distinct heterodimers of gp130 with either leukemia inhibiting factor receptor (LIFR) or OSM receptor-beta (OSMR-beta) (Thoma et al. 1994). Former heterodimer between gp130 and LIFR is called type I receptor complex and the latter between gp130 and OSMR-beta is called type II receptor complex. Type I receptor can bind to either OSM or leukemia inhibiting factor, whereas type II receptor has more affinity towards OSM (O’Hara et al. 2003). The binding of OSM to either gp130/OSMR-beta or gp130/LIFR induces the activation of Janus Kinase family members through tyrosine phosphorylation (Tanaka and Miyajima 2003). The activated JAK family members in turn induce the activation of Signal Transduction and Activator of Transcription (STAT) proteins (Schaefer et al. 2000). Alternatively, the activated receptors can also activate mitogen-activated protein kinase (MAPK) pathway (Van Wagoner et al. 2000) and PI3K/AKT pathways (Arita et al. 2008). It was also reported that OSM bring about ligand-induced receptor degradation of gp130, OSMR-beta, and LIFR before enhancing the synthesis of the receptor subunits (Blanchard et al. 2001).

Quantitative Proteomic and Phosphoproteomic Analysis of H37Ra and H37Rv Strains of Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis, accounts for 1.5 million human deaths annually worldwide. Despite efforts to eradicate tuberculosis, it still remains a deadly disease. The two best characterized strains of M. tuberculosis, virulent H37Rv and avirulent H37Ra, provide a unique platform to investigate biochemical and signaling pathways associated with pathogenicity. To delineate the biomolecular dynamics that may account for pathogenicity and attenuation of virulence in M. tuberculosis, we compared the proteome and phosphoproteome profiles of H37Rv and H37Ra strains. Quantitative phosphoproteomic analysis was performed using high-resolution Fourier transform mass spectrometry. Analysis of exponential and stationary phases of these strains resulted in identification and quantitation of 2709 proteins along with 512 phosphorylation sites derived from 257 proteins. In addition to confirming the presence of previously described M. tuberculosis phosphorylated proteins, we identified 265 novel phosphorylation sites. Quantitative proteomic analysis revealed more than five-fold upregulation of proteins belonging to virulence associated type VII bacterial secretion system in H37Rv when compared to those in H37Ra. We also identified 84 proteins, which exhibited changes in phosphorylation levels between the virulent and avirulent strains. Bioinformatics analysis of the proteins altered in their level of expression or phosphorylation revealed enrichment of pathways involved in fatty acid biosynthesis and two-component regulatory system. Our data provides a resource for further exploration of functional differences at molecular level between H37Rv and H37Ra, which will ultimately explain the molecular underpinnings that determine virulence in tuberculosis.

Mosquito-Borne Diseases and Omics: Tissue-Restricted Expression and Alternative Splicing Revealed by Transcriptome Profiling of Anopheles stephensi

Malaria is one of the most debilitating mosquito-borne diseases with high global health burdens. While much of the research on malaria and mosquito-borne diseases is focused on Africa, Southeast Asia accounts for a sizable portion of the global burden of malaria. Moreover, about 50% of the Asian malaria incidence and deaths have been from India. A promising development in this context is that the completion of genome sequence of Anopheles stephensi, a major malaria vector in Asia, offers new opportunities for global health innovation, including the progress in deciphering the vectorial ability of this mosquito species at a molecular level. Moving forward, tissue-based expression profiling would be the next obvious step in understanding gene functions of An. stephensi. We report in this article, to the best of our knowledge, the first in-depth study on tissue-based transcriptomic profile of four important organs (midgut, Malpighian tubules, fat body, and ovary) of adult female An. stephensi mosquitoes. In all, we identified over 20,000 transcripts corresponding to more than 12,000 gene loci from these four tissues. We present and discuss the tissue-based expression profiles of majority of annotated transcripts in An. stephensi genome, and the dynamics of their alternative splicing in these tissues, in this study. The domain-based Gene Ontology analysis of the differentially expressed transcripts in each of the mosquito tissue indicated enrichment of transcripts with proteolytic activity in midgut; transporter activity in Malpighian tubules; cell cycle, DNA replication, and repair activities in ovaries; and oxidoreductase activities in fat body. Tissue-based study of transcript expression and gene functions markedly enhances our understanding of this important malaria vector, and in turn, offers rationales for further studies on vectorial ability and identification of novel molecular targets to intercept malaria transmission.

Identification of Host-Response in Cerebral Malaria Patients Using Quantitative Proteomic Analysis

The objective of this study was to study the altered proteome in the frontal lobe of patients with CM. Unbiased analysis of differentially abundant proteins could lead to identification of host responses against Plasmodium falciparum infection, which will aid in better understanding of the molecular mechanism of pathophysiology in CM.

Mapping Anopheles stephensi midgut proteome using high-resolution mass spectrometry

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”.

Characterization of human pineal gland proteome

The pineal gland is a neuroendocrine gland located at the center of the brain. It is known to regulate various physiological functions in the body through secretion of the neurohormone melatonin. Comprehensive characterization of the human pineal gland proteome has not been undertaken to date. We employed a high-resolution mass spectrometry-based approach to characterize the proteome of the human pineal gland. A total of 5874 proteins were identified from the human pineal gland in this study. Of these, 5820 proteins were identified from the human pineal gland for the first time. Interestingly, 1136 proteins from the human pineal gland were found to contain a signal peptide domain, which indicates the secretory nature of these proteins. An unbiased global proteomic profile of this biomedically important organ should benefit molecular research to unravel the role of the pineal gland in neuropsychiatric and neurodegenerative diseases.