HUPO Initiatives Relevant to Clinical Proteomics

The past few years have seen a tremendous interest in the potential of proteomics to address unmet needs in biomedicine. Such unmet needs include more effective strategies for early disease detection and monitoring and more effective therapies, in addition to developing a better understanding of disease pathogenesis. Proteomics is particularly suited for investigating biological fluids to identify disease-related alterations and to develop molecular signatures for disease processes. However, much of the effort undertaken in clinical proteomics to date represents either demonstrations of principles or relatively small-scale studies when compared with genomics effort and accomplishments or more pertinently when contrasted with the tremendous untapped potential of clinical proteomics. Clearly, we are in the early stages. What seems to be urgently needed is an organized effort to build a solid foundation for proteomics that includes developing a much needed infrastructure with adequate resources. The Human Proteome Organization (HUPO) is fostering an organized international effort in proteomics that includes initiatives around organ systems and biological fluids that have disease relevance as well as development of proteomics resources. The past few years have seen a tremendous interest in the potential of proteomics to address unmet needs in biomedicine. Such unmet needs include more effective strategies for early disease detection and monitoring and more effective therapies, in addition to developing a better understanding of disease pathogenesis. Proteomics is particularly suited for investigating biological fluids to identify disease-related alterations and to develop molecular signatures for disease processes. However, much of the effort undertaken in clinical proteomics to date represents either demonstrations of principles or relatively small-scale studies when compared with genomics effort and accomplishments or more pertinently when contrasted with the tremendous untapped potential of clinical proteomics. Clearly, we are in the early stages. What seems to be urgently needed is an organized effort to build a solid foundation for proteomics that includes developing a much needed infrastructure with adequate resources. The Human Proteome Organization (HUPO) is fostering an organized international effort in proteomics that includes initiatives around organ systems and biological fluids that have disease relevance as well as development of proteomics resources. Uncovering the wide range of protein alterations in disease states holds much promise for elucidating disease pathogenesis and for developing novel diagnostics and therapeutics (1Hanash S. Disease proteomics. . Nature. 2003; 422: 226-232Google Scholar). To facilitate disease-related discoveries, it would be highly relevant to develop a knowledge base of the normal human proteome. Given the enormous complexity of the human proteome, it stands to reason that no individual institution, be it public or private, would have the needed resources to tackle the human proteome single handedly. The Human Proteome Organization (HUPO, 1The abbreviations used are: HUPO, Human Proteome Organization; PSI, Proteomics Standards Initiative; PPP, Plasma Proteome Project; LPP, Liver Proteome Project; BPP, Brain Proteome Project; DCC, data collection center. www. HUPO. org) came into existence to help identify needed resources and determine achievable objectives to further our understanding of the human proteome. HUPO’s stated mission is to consolidate national and regional proteome organizations into a worldwide organization; to engage in scientific and educational activities to encourage the spread of proteomics technologies; and to disseminate knowledge pertaining to the human proteome and that of model organisms. To help define needed resources and prioritize objectives, HUPO has organized numerous meetings in North America, Asia, and Europe, with participation by government and industry representatives and academicians. As a result, a number of international initiatives have been formulated with goals to develop resources for proteomics and to initiate projects around organ systems and biological fluids that have disease relevance. There is a substantial need for informatics resources for practically every aspect of proteomics (2Boguski M. S. McIntosh M. W. Biomedical informatics for proteomics. . Nature. 2003; 422: 233-237Google Scholar). The current approaches to the analysis of protein data are highly informal and nonstandardized. An important informatics-related effort that HUPO is involved in is aimed at developing and adopting standardized approaches that facilitate analysis of proteomics data generated by different laboratories. The HUPO Proteomics Standards Initiative (PSI) was launched with the aim of defining community standards for data representation in proteomics to facilitate data comparison, exchange, and validation (psidev. sourcefourge. net) (3Orchard S. Zhu W. Julian R. K. J. Hermjakob H. Apweiler R. Further advances in the development of a data interchange standard for proteomics data. . Proteomics. 2003; 3: 2065-2066Google Scholar). The initial focus has been on protein-protein interaction data and mass spectrometry data. Although a number of studies have been undertaken to define protein interaction networks by different groups, data integration from different studies is hampered by the fragmentation of the data that resides in various databases and in different formats. The HUPO PSI, with the support of major protein interaction data providers, has proposed a community standard data model for the representation and exchange of protein interaction data. The development of common standards for data exchange in the field of mass spectrometry as well as for other fields of proteomics are in progress Table I. Hupo informatics efforts will extend to other aspects of proteomics such as quantitative protein expression analysis. They will also address the needs of proteome projects that target organ systems and biological fluids, with respect to data collection, storage, and dissemination (Fig. 1). Table ICurrent agenda for the PSIA. Mass spectrometry1. Controlled vocabulary for m/z data exchange2. Converters to convert supported vendor proprietary peaklist formats toa. Standard XML formatb. Standard XML format for search engine results3. Search engines to be supported4. XML schema5. Tools: navigation, visulization6. How to adapt PSI-MS standard data formats7. PSI-MS data formats and MIAPE data modelB. Molecular interactions1. PSI MI XML schema2. Controlled vocabulariesa. Procedure for maintenance of controlled vocabulariesb. Controlled vocabulary for external databases3. Unified identifiers for interactors4. Data exchange optionsC. Integrating proteomics workflows1. Sample preparation—coordination with MGED2. 2D gel electrophoresis—data capture, data storage3. Modeling columns and other separation techniques4. Integrating mass spectrometry methods and results5. Supporting bioinformatics analyses6. Ontology development Open table in a new tab An important resource-related initiative that HUPO is engaged in is the development of antibodies at the necessary scale for proteome-wide investigations. Such a collection of antibodies would allow for example production of antibody microarrays that assay the proteins expressed in a tissue or contained in a biological fluid. The large collection of antibodies, which will be available to researchers, should also facilitate investigations of individual proteins, given that most proteins encoded in the genome do not have corresponding antibodies. The plans under development consist of initially making antibodies directed against the proteins identified as part of the HUPO organ system and biological fluids proteome projects described below and eventually making antibodies to all the proteins encoded for in the human genome. Two major execution sites for antibody production will be located in Europe and in China, and additional facilities are currently being explored. A piloting phase for antibody production in China has already begun and has yielded to date some 1, 000 antibodies. Much effort has been devoted to identifying a limited number of organ systems and biological fluids that would be the focus of projects to characterize in depth their protein constituents (4Service R. F. Proteomics. Public projects gear up to chart the protein landscape. . Science. 2003; 302: 1316-1318Google Scholar). The choice is based on scientific and medical relevance as well as interest on the part of governments, industry, and funding organizations to commit the necessary funds for execution of the projects. The effort involved in these projects represents an ambitious undertaking that is intended to push the limits of proteomics technologies through a cooperative international effort. These targeted proteome projects will be executed in phases commensurate with the availability of funding and the capabilities of available technologies. HUPO currently has one biological fluid project, namely the Plasma Proteome Project (PPP), and two organ system projects, namely the Liver Proteome Project (LPP) and the Brain Proteome Project (BPP). Each of these projects has been the focus of numerous activities. The first proteome project to be implemented is the PPP, which is currently in its piloting phase. The scientific objectives of the PPP include 1) comprehensive analysis of plasma protein constituents in normal humans in large cohorts of subjects; 2) determination of the extent of variation in plasma proteins within populations in various countries and across various populations from around the world; and 3) identification of biological sources of variation within individuals over time and assessment of the effect of age, sex, diet, and lifestyle, as well as common medications and common diseases. Before the execution of the project would begin on the envisioned scale, a pilot phase is being undertaken. The characterization of plasma proteins is challenging because of the vast dynamic range of protein abundance. The objectives of the pilot phase include a comparison of a broad range of technology platforms for the characterization of proteins in human plasma and serum, in particular low-abundance proteins, thus laying the groundwork for future studies of circulating proteins in health and disease. The pilot phase will allow HUPO to objectively assess the resolution, sensitivity, time involved, costs, and volumes of sample required with various technologies. Another objective is to clarify the influence of technical variables in specimen collection, handling, and storage. There is also a need to assess the merits and feasibility of depleting the most-abundant plasma proteins and the need for additives for protein stability. To this effect, standardized samples have been distributed to some 50 laboratories throughout the world. A breakdown of the various technologies and strategies implemented by participating laboratories is shown in Table 1I. Another objective is to begin to establish international collaborations for later-phase characterization of the normal human plasma proteome in various ethnic groups. Table IIStrategies for serum/plasma protein analysis being tested in the pilot phaseA. Sample preparation strategiesEnrichment for low-abundance proteins (fractionation, affinity capture…) Protein taggingB. Profiling technologiesSeparation based2D/MALDILC/MSNonseparation basedProtein microarraysDirect analysis by mass spectrometryC. Protein detection strategiesStaining, flourescence…Immunee-based assays Open table in a new tab Funds have been raised by HUPO, with equal contributions from industry and from the National Institutes of Health in the United States, for the coordination of the piloting phase and for the award of modest funds for specific activities deemed highly relevant to the pilot phase to some of the participating groups. The pilot phase is expected to end in mid-2004. HUPO is planning to organize a meeting at the completion of the pilot phase to review the findings resulting from the work undertaken by participating laboratories, which is expected to result in a database of identified proteins in serum and plasma and in publications in scientific journals. The technologies that would be proven to have the greatest merit will be utilized using standardized protocols to achieve the scientific objectives of the project. Although currently the biological fluids envisioned are limited to serum and plasma, there is also interest to consider other related biological fluids such as saliva and urine. The LPP has been the subject of meetings in Beijing, Bethesda, and Montreal to formalize the objectives of the project, to assess the availability of needed resources, and to identify participating countries and laboratories (5Cyranoski D. China takes centre stage for liver proteome. . Nature. 2003; 425: 441Google Scholar). Although the plasma presents challenges related to the vast dynamic range of protein abundance, it is far less complex than an organ such as the liver, which includes numerous cell types and thus presents much greater challenges with respect to sampling, storage, and distribution. An important consideration in the planning for the LPP is the availability of tissue samples for distribution as well as the feasibility of preparing purified liver cell populations for analysis. The scientific objectives of the LPP are outlined in Table III. The pilot phase for the LPP consists of analysis of a limited number of liver tissue samples and cultured cells to determine feasibility, as in the case of the PPP. The importance of the LPP is demonstrated by the decision of the government of China to become a major participant in the project with the allocation of some 30 million to the pilot phase of the LPP and a projected contribution of some 200 million for the execution phase. Other countries with important contributions to the project include France, Canada, and the United States. Table IIIScientific objectives of the LPP1. Collection and banking of human liver tissue specimens2. Characterization of the protein expression profile of human liver3. Elucidation of post-translational modifications of liver proteins4. Construction of protein interaction maps of human liver5. Localization of human liver proteins in cellular compartments6. Development of an antibody bank for human liver proteins7. Development of an ORF bank for human liver proteins8. Studies of liver disease with a focus on hepatitis, liver cancer, and related pathologies Open table in a new tab The BPP has been the subject of several workshops to define scientific objectives and to develop aims for a pilot phase, along similar lines as the LPP (6Meyer H. E. Klose J. Hamacher M. HBPP and the pursuit of standardisation. . Lancet Neurol. 2003; 2: 657-658Google Scholar). A planning committee has been constituted to formally organize the project, with Germany and the German Proteome Organization playing a major role at the present time, with active participation by the National Institutes of Health in the United States and several other organizations. For example, a Brain Proteomics workshop was organized jointly with the National Institute of Neurological Diseases and Stroke in Washington, DC in December, 2002. The goals were to engage scientists from various disciplines of proteomics and neuroscience to address issues specific to the brain proteome and to identify the opportunities and requirements for the effective use of proteomics in understanding function and diseases of the nervous system. The merits of characterizing all brain proteins as opposed to emphasis on specific compartments such as the synaptosome or on functionally defined sets of proteins that may be involved in signaling or G protein-coupled receptors, etc. , are being considered. There is also interest in the comparative analyses of model organisms and human. As for the Human Liver Project, the development of resources including cDNA clones, monoclonal antibodies, and tissue repositories are an important component of the project. Several workshops are being planned in 2004 to finalize the organization of the project. Plans for informatics support for the HUPO projects are currently being developed with active participation of the European Bioinformatics Institute and bioinformaticians. A proposed infrastructure for collecting data for the various projects, once they reach the execution phase, which is under consideration, is shown in Fig. 1 and Table IV. A three-tiered structure consisting of 1) laboratories generating data, 2) data collection centers (DCCs) performing quality control and file validation, and 3) central repository of the validated data for public access is being considered. Table IVA summary of the respective roles of the DCCs and the EBIDCC functionsCentral repository functions1. Data collection1. Central proteomics data repository for all HUPO tissue groups2. Conversion to an XML format, complying with the schema shared by all tissue groups (from EBI) 2. Data access interfaces and data distribution3. Synchronize data, in XML, between DCCs3. Coordination of the development of PSI standards4. Quality control4. Coordination of the development of proteomics XML schema integrated with PSI standards5. Morror site of PRIDE5. Providing tools, such as Pedro and peak list converters6. Services for data analyses Open table in a new tab In addition to meetings and workshops dedicated to specific initiatives, HUPO has launched an annual proteomics world congress that is currently in its third year. HUPO’s first world congress took place in Versailles, France in November, 2002 and attracted some 1, 000 participants. The second congress took place in October, 2003 in Montreal, Canada and attracted some 2, 500 participants. This year’s congress will take place in Beijing, China, October 25–28, 2004, with numerous sessions related to medical proteomics. The congress will be preceded by an educational program and a series of workshops dedicated to the HUPO initiatives. Although HUPO has been in existence for some 3 years only, substantial progress has been made in forging partnerships between the public and the private sectors and in uniting efforts in different countries to help build a solid foundation for proteomics that help address unmet needs in biomedicine.