Renu Goel

Human Protein Reference Database—2009 update

Human Protein Reference Database (HPRD—http://www.hprd.org/), initially described in 2003, is a database of curated proteomic information pertaining to human proteins. We have recently added a number of new features in HPRD. These include PhosphoMotif Finder, which allows users to find the presence of over 320 experimentally verified phosphorylation motifs in proteins of interest. Another new feature is a protein distributed annotation system—Human Proteinpedia (http://www.humanproteinpedia.org/)—through which laboratories can submit their data, which is mapped onto protein entries in HPRD. Over 75 laboratories involved in proteomics research have already participated in this effort by submitting data for over 15 000 human proteins. The submitted data includes mass spectrometry and protein microarray-derived data, among other data types. Finally, HPRD is also linked to a compendium of human signaling pathways developed by our group, NetPath (http://www.netpath.org/), which currently contains annotations for several cancer and immune signaling pathways. Since the last update, more than 5500 new protein sequences have been added, making HPRD a comprehensive resource for studying the human proteome.

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.

NetPath: a public resource of curated signal transduction pathways.

We have developed NetPath as a resource of curated human signaling pathways. As an initial step, NetPath provides detailed maps of a number of immune signaling pathways, which include approximately 1,600 reactions annotated from the literature and more than 2,800 instances of transcriptionally regulated genes - all linked to over 5,500 published articles. We anticipate NetPath to become a consolidated resource for human signaling pathways that should enable systems biology approaches.

A Compendium of Potential Biomarkers of Pancreatic Cancer

Akhilesh Pandey and colleagues describe a compendium of potential biomarkers that can be systematically validated by the pancreatic cancer community.

Construction of human activity-based phosphorylation networks.

The landscape of human phosphorylation networks has not been systematically explored, representing vast, unchartered territories within cellular signaling networks. Although a large number of in vivo phosphorylated residues have been identified by mass spectrometry (MS)‐based approaches, assigning the upstream kinases to these residues requires biochemical analysis of kinase‐substrate relationships (KSRs). Here, we developed a new strategy, called CEASAR, based on functional protein microarrays and bioinformatics to experimentally identify substrates for 289 unique kinases, resulting in 3656 high‐quality KSRs. We then generated consensus phosphorylation motifs for each of the kinases and integrated this information, along with information about in vivo phosphorylation sites determined by MS, to construct a high‐resolution map of phosphorylation networks that connects 230 kinases to 2591 in vivo phosphorylation sites in 652 substrates. The value of this data set is demonstrated through the discovery of a new role for PKA downstream of Btk (Brutons tyrosine kinase) during B‐cell receptor signaling. Overall, these studies provide global insights into kinase‐mediated signaling pathways and promise to advance our understanding of cellular signaling processes in humans.

A comprehensive map of the human urinary proteome.

The study of the human urinary proteome has the potential to offer significant insights into normal physiology as well as disease pathology. The information obtained from such studies could be applied to the diagnosis of various diseases. The high sensitivity, resolution, and mass accuracy of the latest generation of mass spectrometers provides an opportunity to accurately catalog the proteins present in human urine, including those present at low levels. To this end, we carried out a comprehensive analysis of human urinary proteome from healthy individuals using high-resolution Fourier transform mass spectrometry. Importantly, we used the Orbitrap for detecting ions in both MS (resolution 60 000) and MS/MS (resolution 15 000) modes. To increase the depth of our analysis, we characterized both unfractionated as well as lectin-enriched proteins in our experiments. In all, we identified 1,823 proteins with less than 1% false discovery rate, of which 671 proteins have not previously been reported as constituents of human urine. This data set should serve as a comprehensive reference list for future studies aimed at identification and characterization of urinary biomarkers for various diseases.

Human Protein Reference Database and Human Proteinpedia as resources for phosphoproteome analysis

Human Protein Reference Database (HPRD) is a rich resource of experimentally proven features of human proteins. Protein information in HPRD includes protein-protein interactions, post-translational modifications, enzyme/substrate relationships, disease associations, tissue expression, and subcellular localization of human proteins. Although, protein-protein interaction data from HPRD has been widely used by the scientific community, its phosphoproteome data has not been exploited to its full potential. HPRD is one of the largest documentations of human phosphoproteins in the public domain. Currently, phosphorylation data in HPRD comprises of 95,016 phosphosites mapped on to 13,041 proteins. Additionally, enzyme-substrate reactions responsible for 5930 phosphorylation events were also documented. Significant improvements in technologies and high-throughput platforms in biomedical investigations led to an exponential increase of biological data and phosphoproteomic data in recent years. Human Proteinpedia, a community annotation portal developed by us, has also contributed to the significant increase in phosphoproteomic data in HPRD. A large number of phosphorylation events have been mapped on to reference sequences available in HPRD and Human Proteinpedia along with associated protein features. This will provide a platform for systems biology approaches to determine the role of protein phosphorylation in protein function, cell signaling, biological processes and their implication in human diseases. This review aims to provide a composite view of phosphoproteomic data pertaining to human proteins in HPRD and Human Proteinpedia.

Temporal Profiling of the Secretome during Adipogenesis in Humans

Adipose tissue plays a key role as a fat-storage depot and as an endocrine organ. Although mouse adipogenesis has been studied extensively, limited studies have been conducted to characterize this process in humans. We carried out a temporal proteomic analysis to interrogate the dynamic changes in the secretome of primary human preadipocytes as they differentiate into mature adipocytes. Using iTRAQ-based quantitative proteomics, we identified and quantified 420 proteins from the secretome of differentiated human adipocytes. Our results revealed that the majority of proteins showed differential expression during the course of differentiation. In addition to adipokines known to be differentially secreted in the course of adipocyte differentiation, we identified a number of proteins whose dynamic expression in this process has not been previously documented. They include collagen triple helix repeat containing 1, cytokine receptor-like factor 1, glypican-1, hepatoma-derived growth factor, SPARC related modular calcium binding protein 1, SPOCK 1, and sushi repeat-containing protein. A bioinformatics analysis using Human Protein Reference Database and Human Proteinpedia revealed that of the 420 proteins identified, 164 proteins possess signal peptides and 148 proteins are localized to the extracellular compartment. Additionally, we employed antibody arrays to quantify changes in the levels of 182 adipokines during human adipogenesis. This is the first large-scale quantitative proteomic study that combines two platforms, mass spectrometry and antibody arrays, to analyze the changes in the secretome during the course of adipogenesis in humans.

Human Proteinpedia: A unified discovery resource for proteomics research

Sharing proteomic data with the biomedical community through a unified proteomic resource, especially in the context of individual proteins, is a challenging prospect. We have developed a community portal, designated as Human Proteinpedia (http://www.humanproteinpedia.org/), for sharing both unpublished and published human proteomic data through the use of a distributed annotation system designed specifically for this purpose. This system allows laboratories to contribute and maintain protein annotations, which are also mapped to the corresponding proteins through the Human Protein Reference Database (HPRD; http://www.hprd.org/). Thus, it is possible to visualize data pertaining to experimentally validated posttranslational modifications (PTMs), protein isoforms, protein–protein interactions (PPIs), tissue expression, expression in cell lines, subcellular localization and enzyme substrates in the context of individual proteins. With enthusiastic participation of the proteomics community, the past 15 months have witnessed data contributions from more than 75 labs around the world including 2710 distinct experiments, >1.9 million peptides, >4.8 million MS/MS spectra, 150 368 protein expression annotations, 17 410 PTMs, 34 624 PPIs and 2906 subcellular localization annotations. Human Proteinpedia should serve as an integrated platform to store, integrate and disseminate such proteomic data and is inching towards evolving into a unified human proteomics resource.

Human protein reference database and human proteinpedia as discovery resources for molecular biotechnology.

In the recent years, research in molecular biotechnology has transformed from being small scale studies targeted at a single or a small set of molecule(s) into a combination of high throughput discovery platforms and extensive validations. Such a discovery platform provided an unbiased approach which resulted in the identification of several novel genetic and protein biomarkers. High throughput nature of these investigations coupled with higher sensitivity and specificity of Next Generation technologies provided qualitatively and quantitatively richer biological data. These developments have also revolutionized biological research and speed of data generation. However, it is becoming difficult for individual investigators to directly benefit from this data because they are not easily accessible. Data resources became necessary to assimilate, store and disseminate information that could allow future discoveries. We have developed two resources—Human Protein Reference Database (HPRD) and Human Proteinpedia, which integrate knowledge relevant to human proteins. A number of protein features including protein–protein interactions, post-translational modifications, subcellular localization, and tissue expression, which have been studied using different strategies were incorporated in these databases. Human Proteinpedia also provides a portal for community participation to annotate and share proteomic data and uses HPRD as the scaffold for data processing. Proteomic investigators can even share unpublished data in Human Proteinpedia, which provides a meaningful platform for data sharing. As proteomic information reflects a direct view of cellular systems, proteomics is expected to complement other areas of biology such as genomics, transcriptomics, molecular biology, cloning, and classical genetics in understanding the relationships among multiple facets of biological systems.