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.

Host response profile of human brain proteome in toxoplasma encephalitis co-infected with HIV.

BACKGROUND: Toxoplasma encephalitis is caused by the opportunistic protozoan parasite Toxoplasma gondii. Primary infection with T. gondii in immunocompetent individuals remains largely asymptomatic. In contrast, in immunocompromised individuals, reactivation of the parasite results in severe complications and mortality. Molecular changes at the protein level in the host central nervous system and proteins associated with pathogenesis of toxoplasma encephalitis are largely unexplored. We used a global quantitative proteomic strategy to identify differentially regulated proteins and affected molecular networks in the human host during T. gondii infection with HIV co-infection. RESULTS: We identified 3,496 proteins out of which 607 proteins were differentially expressed (≥1.5-fold) when frontal lobe of the brain from patients diagnosed with toxoplasma encephalitis was compared to control brain tissues. We validated differential expression of 3 proteins through immunohistochemistry, which was confirmed to be consistent with mass spectrometry analysis. Pathway analysis of differentially expressed proteins indicated deregulation of several pathways involved in antigen processing, immune response, neuronal growth, neurotransmitter transport and energy metabolism. CONCLUSIONS: Global quantitative proteomic approach adopted in this study generated a comparative proteome profile of brain tissues from toxoplasma encephalitis patients co-infected with HIV. Differentially expressed proteins include previously reported and several new proteins in the context of T. gondii and HIV infection, which can be further investigated. Molecular pathways identified to be associated with the disease should enhance our understanding of pathogenesis in toxoplasma encephalitis.

Quantitative proteomics for identifying biomarkers for Rabies.

INTRODUCTION: Rabies is a fatal acute viral disease of the central nervous system, which is a serious public health problem in Asian and African countries. Based on the clinical presentation, rabies can be classified into encephalitic (furious) or paralytic (numb) rabies. Early diagnosis of this disease is particularly important as rabies is invariably fatal if adequate post exposure prophylaxis is not administered immediately following the bite. METHODS: In this study, we carried out a quantitative proteomic analysis of the human brain tissue from cases of encephalitic and paralytic rabies along with normal human brain tissues using an 8-plex isobaric tags for relative and absolute quantification (iTRAQ) strategy. RESULTS AND CONCLUSION: We identified 402 proteins, of which a number of proteins were differentially expressed between encephalitic and paralytic rabies, including several novel proteins. The differentially expressed molecules included karyopherin alpha 4 (KPNA4), which was overexpressed only in paralytic rabies, calcium calmodulin dependent kinase 2 alpha (CAMK2A), which was upregulated in paralytic rabies group and glutamate ammonia ligase (GLUL), which was overexpressed in paralytic as well as encephalitic rabies. We validated two of the upregulated molecules, GLUL and CAMK2A, by dot blot assays and further validated CAMK2A by immunohistochemistry. These molecules need to be further investigated in body fluids such as cerebrospinal fluid in a larger cohort of rabies cases to determine their potential use as antemortem diagnostic biomarkers in rabies. This is the first study to systematically profile clinical subtypes of human rabies using an iTRAQ quantitative proteomics approach.

Proteogenomic analysis of Candida glabrata using high resolution mass spectrometry.

Candida glabrata is a common opportunistic human pathogen leading to significant mortality in immunosuppressed and immunodeficient individuals. We carried out proteomic analysis of C. glabrata using high resolution Fourier transform mass spectrometry with MS resolution of 60,000 and MS/MS resolution of 7500. On the basis of 32,453 unique peptides identified from 118,815 peptide-spectrum matches, we validated 4421 of the 5283 predicted protein-coding genes (83%) in the C. glabrata genome. Further, searching the tandem mass spectra against a six frame translated genome database of C. glabrata resulted in identification of 11 novel protein coding genes and correction of gene boundaries for 14 predicted gene models. A subset of novel protein-coding genes and corrected gene models were validated at the transcript level by RT-PCR and sequencing. Our study illustrates how proteogenomic analysis enabled by high resolution mass spectrometry can enrich genome annotation and should be an integral part of ongoing genome sequencing and annotation efforts.

RAPID: Resource of Asian Primary Immunodeficiency Diseases.

Availability of a freely accessible, dynamic and integrated database for primary immunodeficiency diseases (PID) is important both for researchers as well as clinicians. To build a PID informational platform and also as a part of action to initiate a network of PID research in Asia, we have constructed a web-based compendium of molecular alterations in PID, named Resource of Asian Primary Immunodeficiency Diseases (RAPID), which is available as a worldwide web resource at http://rapid.rcai.riken.jp/. It hosts information on sequence variations and expression at the mRNA and protein levels of all genes reported to be involved in PID patients. The main objective of this database is to provide detailed information pertaining to genes and proteins involved in primary immunodeficiency diseases along with other relevant information about protein-protein interactions, mouse studies and microarray gene-expression profiles in various organs and cells of the immune system. RAPID also hosts a tool, mutation viewer, to predict deleterious and novel mutations and also to obtain mutation-based 3D structures for PID genes. Thus, information contained in this database should help physicians and other biomedical investigators to further investigate the role of these molecules in PID.

Proteomics-based approach for differentiation of age-related macular degeneration sub-types.

Purpose: Age-related macular degeneration (AMD) is one of the leading causes of irreversible central vision loss in the elderly population. The current study aims to find non-invasive prognostic biomarkers in the urine specimens of the AMD patients. Methods: Peripheral blood and urine samples were collected from 23 controls and 61 AMD patients. Genomic DNA was extracted from the buffy coat of peripheral blood. Allele specific PCR was used to assay SNPs in complement factor H (CFH), complement component 3 (C3). Comparative proteomic analysis of urine samples from early AMD, choroidal neovascular membrane (CNVM), geographic atrophy (GA), and healthy controls was performed using isobaric labelling followed by mass spectrometry. Validation was performed using enzyme-linked immunosorbent assay (ELISA). Results: Comparative proteomic analysis of urine samples identified 751 proteins, of which 383 proteins were found to be differentially expressed in various groups of AMD patients. Gene ontology classification of differentially expressed proteins revealed the majority of them were involved in catalytic functions and binding activities. Pathway analysis showed cell adhesion molecule pathways (CAMs), Complement and coagulation cascades, to be significantly deregulated in AMD. Upon validation by ELISA, SERPINA-1 (Alpha1 antitrypsin), TIMP-1 (Tissue inhibitor of matrix metaloprotease-1), APOA-1 (Apolipoprotein A-1) were significantly over-expressed in AMD (n = 61) patients compared to controls (n = 23). A logistic model of APOA-1 in combination with CFH and C3 polymorphisms predicted the risk of developing AMD with 82% accuracy. Conclusion: This study gives us a preliminary data on non-invasive predictive biomarkers for AMD, which can be further validated in a large cohort and translated for diagnostic use.

Exome sequencing reveals a novel splice site variant in HUWE1 gene in patients with suspected Say-Meyer syndrome.

Say-Meyer syndrome is a rare and clinically heterogeneous syndrome characterized by trigonocephaly, short stature, developmental delay and hypotelorism. Nine patients with this syndrome have been reported thus far although no causative gene has yet been identified. Here, we report two siblings with clinical phenotypes of Say-Meyer syndrome with moderate to severe intellectual disability and autism spectrum disorder. Cytogenetics and array-based comparative genomic hybridization did not reveal any chromosome abnormalities or copy number alterations. Exome sequencing of the patients revealed a novel X-linked recessive splice acceptor site variant c.145-2A > G in intron 5 of HUWE1 gene in both affected siblings. RT-PCR and sequencing revealed the use of an alternate cryptic splice acceptor site downstream, which led to deletion of six nucleotides resulting loss of two amino acids p.(Cys49-Glu50del) in HUWE1 protein. Deletion of these two amino acids, which are located in a highly conserved region, is predicted to be deleterious and quite likely to affect the function of HUWE1 protein. This is the first report of a potential candidate gene mutation for Say-Meyer syndrome, which was initially described four decades ago.

Next-Generation Sequencing Reveals Novel Mutations in X-linked Intellectual Disability.

Robust diagnostics for many human genetic disorders are much needed in the pursuit of global personalized medicine. Next-generation sequencing now offers new promise for biomarker and diagnostic discovery, in developed as well as resource-limited countries. In this broader global health context, X-linked intellectual disability (XLID) is an inherited genetic disorder that is associated with a range of phenotypes impacting societies in both developed and developing countries. Although intellectual disability arises due to diverse causes, a substantial proportion is caused by genomic alterations. Studies have identified causal XLID genomic alterations in more than 100 protein-coding genes located on the X-chromosome. However, the causes for a substantial number of intellectual disability and associated phenotypes still remain unknown. Identification of causative genes and novel mutations will help in early diagnosis as well as genetic counseling of families. Advent of next-generation sequencing methods has accelerated the discovery of new genes involved in mental health disorders. In this study, we analyzed the exomes of three families from India with nonsyndromic XLID comprising seven affected individuals. The affected individuals had varying degrees of intellectual disability, microcephaly, and delayed motor and language milestones. We identified potential causal variants in three XLID genes, including PAK3 (V294M), CASK (complex structural variant), and MECP2 (P354T). Our findings reported in this study extend the spectrum of mutations and phenotypes associated with XLID, and calls for further studies of intellectual disability and mental health disorders with use of next-generation sequencing technologies.

Characterization of host response to Cryptococcus neoformans through quantitative proteomic analysis of cryptococcal meningitis co-infected with HIV.

Cryptococcal meningitis is the most common opportunistic fungal infection causing morbidity and mortality (>60%) in HIV-associated immunocompromised individuals caused by Cryptococcus neoformans. Molecular mechanisms of cryptococcal infection in brain have been studied using experimental animal models and cell lines. There are limited studies for the molecular understanding of cryptococcal meningitis in human brain. The proteins involved in the process of invasion and infection in human brain still remains obscure. To this end we carried out mass spectrometry-based quantitative proteomics of frontal lobe brain tissues from cryptococcal meningitis patients and controls to identify host proteins that are associated with the pathogenesis of cryptococcal meningitis. We identified 317 proteins to be differentially expressed (≥2-fold) from a total of 3423 human proteins. We found proteins involved in immune response and signal transduction to be differentially expressed in response to cryptococcal infection in human brain. Immune response proteins including complement factors, major histocompatibility proteins, proteins previously known to be involved in fungal invasion to brain such as caveolin 1 and actin were identified to be differentially expressed in cryptococcal meningitis brain tissues co-infected with HIV. We also validated the expression status of 5 proteins using immunohistochemistry. Overexpression of major histocompatibility complexes, class I, B (HLA-B), actin alpha 2 smooth muscle aorta (ACTA2) and caveolin 1 (CAV1) and downregulation of peripheral myelin protein 2 (PMP2) and alpha crystallin B chain (CRYAB) in cryptococcal meningitis were confirmed by IHC-based validation experiments. This study provides the brain proteome profile of cryptococcal meningitis co-infected with HIV for a better understanding of the host response associated with the disease.

Phosphoproteome of Cryptococcus neoformans.

Cryptococcus neoformans is an encapsulated pathogenic yeast, which causes life threatening meningitis in immunocompromised individuals. C. neoformans var. grubii is the most prevalent and virulent form among the two varieties of C. neoformans - C. neoformans var. grubii and C. neoformans var. neoformans. The virulence of C. neoformans is mainly conferred by its capsule and melanin. cAMP dependent PKA-induced phosphorylation events are reported to be associated with the expression of these virulence traits, which highlights the importance of phosphoproteins in virulence and infection. Therefore, we performed global profiling of phosphoproteome of C. neoformans to enable a better understanding of molecular regulation of its virulence and pathogenesis. High resolution mass spectrometry of TiO2 enriched phosphopeptides from C. neoformans var. grubii grown in culture led to the identification of 1089 phosphopeptides derived from 648 proteins including about 45 kinases. Motif enrichment analysis revealed that most CDK family substrates were found to be phosphorylated. This indicates that cyclin-dependent kinases were among the active kinases in the pathogen in culture. These studies provide a framework for understanding virulence mechanisms in the context of signalling pathways in pathogenic yeast. This article is part of a Special Issue entitled: Trends in Microbial Proteomics. BIOLOGICAL SIGNIFICANCE: C. neoformans is a pathogenic yeast responsible for cryptococcal meningitis. Melanin and polysaccharide capsule have been established as some of the key virulence factors that play a major role in the pathogenesis of C. neoformans. Recent studies have shown the role of kinase mediated signalling pathways in governing biosynthesis of these virulence factors. This study revealed 1540 phosphorylation sites in 648 proteins providing a comprehensive view of phosphoproteins in C. neoformans. This should serve as a useful resource to explore activated signalling pathways in C. neoformans and their association with its virulence and pathogenesis.

Transcriptomic Profiling of Medial Temporal Lobe Epilepsy.

Epilepsy is one of the most prevalent neurological disorders affecting ~1% of the population. Medial temporal lobe epilepsy (MTLE) is the most frequent type of epilepsy observed in adults who do not respond to pharmacological treatment. The reason for intractability in these patients has not been systematically studied. Further, no markers are available that can predict the subset of patients who will not respond to pharmacotherapy. To identify potential biomarkers of epileptogenicity, we compared the mRNA profiles of surgically resected tissue from seizure zones with non-seizure zones from cases of intractable MTLE. We identified 413 genes that exhibited ≥2-fold change that were statistically significant across these two groups. Several of these differentially expressed genes have not been previously described in the context of MTLE including claudin 11 (CLDN11) and bone morphogenetic protein receptor, type IB (BMPR1B). In addition, we found significant downregulation of a subset of gamma-aminobutyric acid (GABA) associated genes. We also identified molecules such as BACH2 and ADAMTS15, which are already known to be associated with epilepsy. We validated one upregulated molecule, serine/threonine kinase 31 (STK31) and one downregulated molecule, SMARCA4, by immunohistochemical labeling of tissue sections. These molecules need to be further confirmed in large-scale studies to determine their potential use as diagnostic as well as prognostic markers in intractable MTLE.

Identifying targets of miR-143 using a SILAC-based proteomic approach.

Although the targets of most miRNAs have not been experimentally identified, microRNAs (miRNAs) have begun to be extensively characterized in physiological, developmental and disease-related contexts in recent years. Thus far, mainly computational approaches have been employed to predict potential targets for the large majority of miRNAs. Although miRNAs exert a major influence on the efficiency of translation of their targets in animals, most studies describing experimental identification of miRNA target genes are based on detection of altered mRNA levels. miR-143 is a miRNA involved in tumorigenesis in multiple types of cancer, smooth muscle cell fate and adipocyte differentiation. Only a few miR-143 targets are experimentally verified, so we employed a SILAC-based quantitative proteomic strategy to systematically identify potential targets of miR-143. In total, we identified >1200 proteins from MiaPaCa2 pancreatic cancer cells, of which 93 proteins were downregulated >2-fold in miR-143 mimic transfected cells as compared to controls. Validation of 34 of these candidate targets in luciferase assays showed that 10 of them were likely direct targets of miR-143. Importantly, we also carried out gene expression profiling of the same cells and observed that the majority of the candidate targets identified by proteomics did not show a concomitant decrease in mRNA levels confirming that miRNAs affect the expression of most targets through translational inhibition. Our study clearly demonstrates that quantitative proteomic approaches are important and necessary for identifying miRNA targets.