The Moscow HUPO Human Proteome Project Workshop

A workshop was convened in Moscow by the Human Proteome Organization on March 2009 to review the current status of the Human Proteome Project (HPP) 1The abbreviations used are:HPPHuman Proteome ProjectPTMpost-translational modificationMRMmultiple reaction monitoringAQUAAbsolute QuantitationPSAQprotein standard absolute quantification. 1The abbreviations used are:HPPHuman Proteome ProjectPTMpost-translational modificationMRMmultiple reaction monitoringAQUAAbsolute QuantitationPSAQprotein standard absolute quantification. and to discuss how this project could be most effectively launched. Presentations were delivered by representatives from 8 countries, including Russia, Canada, China, France, Japan, Korea, Sweden, the USA as well as the European Commission. Human Proteome Project post-translational modification multiple reaction monitoring Absolute Quantitation protein standard absolute quantification. Human Proteome Project post-translational modification multiple reaction monitoring Absolute Quantitation protein standard absolute quantification. A Russian focus on chromosome 18 was covered by Prof. Alexander Archakov with a proposal to use atomic force microscopy and the iterative irreversible association of biomolecules onto chemically active surfaces to characterize proteins at concentrations below 10−12 m. Support for the Russian contribution to the Human Proteome Project was extended by the Deputy Minister of Education and Science of the Russian Federation, Alexander Khlunov, and Anatoly Grigoriev, Vice-President of the Russian Academy of Sciences. Dr. Grigoriev proposed to extend a disease-centered paradigm to the variability of the proteome in the normal healthy person with support indicated by Alexander Gintsburg, Vice-President of the Russian Academy of Medical Sciences and a Member of the Presidential Council for Science and High Technologies, Academician Konstantin Skryabin. He indicated that Russia has already created technical and human resources for large-scale international projects. Workshop presentations and discussions revolved around a white paper of the HPP, put together during a Barbados workshop in January 2008 and finalized in August 2008. The vision of the white paper comprised three research engines, interfaced to each other at the bioinformatics and computational biology layer. The first engine defines the complete proteome through a mapping with antibodies. In this regard, Prof. F. Ponten (Uppsala University) revealed progress on the Human Protein Atlas Project. He also illustrated the clinical relevance of a systematic antibody mapping of the proteome. The core of the second engine depends upon mass spectrometry, and here many speakers shared the opinion of Dr. S. Hanash (Fred Hutchinson Cancer Research Center, USA) that the technology is already accurate enough to deliver for the medicinal sector. For example, within the International Disease Biomarker Project the cancer biomarkers requirements include risk assessment, early detection, molecular classification, and disease monitoring. However, Prof. R. Bradshaw (University of California, San Francisco) drew attention to the lack of biomarker identification to date and the fact that few if any validated biomarkers have resulted from proteomics experiments (1Zolg W. The proteomic search for diagnostic biomarkers: lost in translation?.Mol. Cell. Proteomics. 2006; 5: 1720-1726Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). He noted that technology may not yet be able to detect biomarkers in complex samples such as plasma, or worse, there may be no useful biomarkers to detect. On the positive side, Dr. N. Taniguchi from Osaka University illustrated the diagnostic power of core fucosylation of N-glycans using mass spectrometry and the fucose-binding Aleuria aurantia lectin. Even analyzing the proteins within the normal range of mass spectrometry detection (haptoglobin, alpha-fetoprotein, etc.), it was possible to unravel the differences associated with emphysema, pancreatic cancer, hepatoma, and liver cirrhosis by focusing on fucose modifications. The progress and already realized clinical applications of glycomics were compared with that of phosphoproteomics. As indicated by Dr. Ralph Bradshaw, even though protein phosphorylations represent a minor component of detectable post-translational modifications (PTMs), the literature is strongly biased toward the description of protein phosphorylation. The general scaffold of how a country can organize its portion of HPP duties in compliance to the joint efforts around the world was presented by the HUPO President Prof. Young Ki-Paik (Yonsei University, Korea). With chromosome 13 as an example, he navigated participants of the workshop through complementary research strategies, which spanned through a starting two-year period of the project with a workflow scheduled for 2010–2018. Again, three aspects of the gene-centric HPP constituted the core of the Korean approach: MS-based, antibody-based, and network-based engines. Strategy one covered multiple reaction monitoring (MRM)-based protein quantification, whereas strategy two also included hybrid antibodies at the protein enrichment stage. The problem of quantification was also an issue of the other reports, starting from the semi-quantitative approach practiced by the Human Liver Proteome Project (HLPP) chosen by Dr. P. Yang (Fudan University) and Absolute Quantitation (AQUA) chosen by KHUPO, and ending with a Protein Standard Absolute Quantification (PSAQ) method, which utilizes full-length isotope-labeled proteins. The latter approach was presented by Prof. Pierre Legrain (Commissariat à l'Energie Atomique, France) to demonstrate its preferences over the conventional protein quantification technologies such as AQUA and Quantification concatamers (QconCAT) for absolute quantification employing concatenated signature peptides of artificial proteins. The difficulty of protein quantification led to an appreciation as to how a gene-centric approach can be used to share the burden of HPP expenses among countries. Whether it is AQUA or PSAQ or any other, a single batch of labeled peptides or proteins may need to be explored by many labs. The price per protein coding gene becomes feasible in the case that the standards are available to many users. Approximate figures can be drawn from the paper by Anderson et al. (2Anderson N.L. Anderson N.G. Pearson T.W. Borchers C.H. Paulovich A.G. Patterson S.D. Gillette M. Aebersold R. Carr S.A. Human proteome detection and quantitation project.Mol. Cell. Proteomics. 2009; 5: 883-886Abstract Full Text Full Text PDF Scopus (172) Google Scholar), who launched the human Proteome Detection and Quantitation (hPDQ) initiative to enable individual researchers to measure defined collections of human proteins in biosamples. The price of isotopic assays of proteotypic peptides was assessed as 50 million USD for 2000 proteins and 250 million USD for all protein-coding genes. Such costs would be prohibitive for any one nation, but since any one nation could be responsible for only one chromosome, the expenses can be shared. This would imply the need for 24 nations with 22 for the autosomes and 2 for the sex chromosomes. In the course of round-table discussions, advances in peptidomic research were overviewed by Prof. V. Ivanov (Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences). The peptidome offers considerable promise for diagnostic purposes, as pathological processes may be linked to reproducible changes in the peptide composition of biofluids. Furthermore, the peptidome may become a source of biologically active peptides, which can be turned into pharmacological compounds (3Ivanov V.T. Yatskin O.N. Peptidomics: a logical sequel to proteomics.Expert. Rev. Proteomics. 2005; 2: 463-473Crossref PubMed Scopus (51) Google Scholar). Projects in human health and disease will likely articulate a protein-centric view of genes as a complement to the gene-centric view of proteins (4Hochstrasser D. Should the human proteome project be gene- or protein-centric?.J. Proteome Res. 2008; 7: 5071Crossref PubMed Scopus (8) Google Scholar). That would be compliant with the disease-related mission of the project. The importance of quantitative analysis in the development of proteomics was emphasized by Prof. R. Bradshaw who indicated, “In proteomics, we generally collect snapshots, whereas in reality the proteome is a movie. To bridge this gap, quantitative measurements are and will be essential and this is the future direction of much of proteomics research. Within two years it will likely become the norm rather than the exception”. In this regard, the recent Clinical Proteomic Technology Assessment for Cancer (CPTAC) study (5Addona T.A. Abbatiello S.E. Schilling B. Skates S.J. Mani D.R. Bunk D.M. Spiegelman C.H. Zimmerman L.J. Ham A.J. Keshishian H. Hall S.C. Allen S. Blackman R.K. Borchers C.H. Buck C. Cardasis H.L. Cusack M.P. Dodder N.G. Gibson B.W. Held J.M. Hiltke T. Jackson A. Johansen E.B. Kinsinger C.R. Li J. Mesri M. Neubert T.A. Niles R.K. Pulsipher T.C. Ransohoff D. Rodriguez H. Rudnick P.A. Smith D. Tabb D.L. Tegeler T.J. Variyath A.M. Vega-Montoto L.J. Wahlander A. Waldemarson S. Wang M. Whiteaker J.R. Zhao L. Anderson N.L. Fisher S.J. Liebler D.C. Paulovich A.G. Regnier F.E. Tempst P. Carr S.A. Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma.Nat. Biotechnol. 2009; 27: 633-641Crossref PubMed Scopus (819) Google Scholar) on the reproducibility of multiple reaction monitoring based measurements of proteins in plasma complements the HUPO test sample study (6Bell A.W. Deutsch E.W. Au C.E. Kearney R.E. Beavis R. Sechi S. Nilsson T. Bergeron J.J.M. HUPO Test Sample Working Group A HUPO test sample study reveals common problems in mass spectrometry-based proteomics.Nat. Methods. 2009; 6: 423-430Crossref PubMed Scopus (259) Google Scholar) to demonstrate reproducibility and accuracy of characterization of test sample proteins. The thesis of “no plasma - no money” (Dr. S. Hanash) was indicated, but whether all of the proteins actually reside in the plasma is unknown. To populate genes with MS spectra, it is necessary to find the preferential residence of desired proteins by antibody mapping of protein localizations as deduced from the predicted protein coding human genes. After that, inspection of a particular cellular subfraction may have a greater chance to access proteins rather than plasma. No dedicated database for the HPP exists today. It was proposed that HPP database construction will be needed under the supervision of HUPO. This database would follow up the gene-centric principle as a convenient scaffold to assemble the data produced by the HPP research engines. The idea behind such a database is to create the global source, which anyone can access for an update, for knowledge that emanates from the HPP. This database will serve as a central warehouse to collect data from the gene-centric databases for antibody and mass spectrometry-based proteomics. We thank Dr. Gil Omenn (University of Michigan), Chair of HUPO Initiatives, for his helpful comments.

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