Introduction

A comprehensive, systematic characterization of circulating proteins in health and disease will greatly facilitate development of biomarkers for prevention, diagnosis, and therapy of cancers and other diseases [1]. Proteomics technologies now permit extensive fractionation of proteins in complex specimens, analysis of peptides by MS, and matching of peptide sequences to protein "hits" through gene and protein databases generated directly and indirectly from the sequencing of the human genome [2, 3], as well as other methods for identifying proteins.

The HUPO, formed in 2001, aims to accelerate the development of the field of proteomics and to stimulate and organize international collaborations in research and education [4]. HUPO has launched major initiatives focused on the plasma, liver, and brain proteomes, proteomics standards and databases, and large-scale antibody production. The plasma proteome is linked with these other initiatives (see Fig. 1).

The long-term scientific goals of the HUPO Plasma Proteome Project (PPP) are (1) comprehensive analysis of the protein constituents of human plasma and serum; (2) identification of biological sources of variation within individuals over time due to physiology (age, sex, menstrual cycle, exercise, stress), pathology (various diseases, special cohorts), and treatments (common medications); and (3) determination of the extent of variation across individuals within populations and across populations due to genetic, nutritional and other factors. The pilot phase aims to (1) compare advantages and limitations of many technology platforms; (2) contrast reference specimens of human plasma (EDTA, heparin, or cit-rate-anticoagulated) and serum in terms of numbers of proteins identified and any interferences with various technology platforms; and (3) create a global, open-source knowledge base/data repository.

The collaborative nature of this Project permitted exploration of many variables and adoption during the study phase of emerging technologies. Planning proceeded expeditiously from the organizing meeting of HUPO in Bethesda in

Serum vs Plasma

Technology Platforms-Separation and Identification

Reference

Serum vs Plasma

Specimens

Participating Labs

HUPO PPP

Development & Validation of Biomarkers

Liver Proteome, Brain Proteome, Antibodies, Protein Standards

Specimens

Participating Labs

HUPO PPP

Development & Validation of Biomarkers

Technology Vendors

Liver Proteome, Brain Proteome, Antibodies, Protein Standards

Fig. 1 Schema showing relationship of HUPO Plasma Proteome Project

(PPP) to other HUPO initiatives and components of the PPP.

April 2002, to the first PPP meeting in Ann Arbor in September 2002, the expression of interest by numerous investigators at the 1st HUPO World Congress on Proteomics in Versailles in November 2002, and then the PPP Workshop for Technical Committees and participating laboratories in Bethesda in July 2003 to launch the pilot phase. PPP reference specimens were prepared and distributed, beginning in September 2003, and first data were submitted, analyzed, and presented at a workshop at the 2nd HUPO World Congress in Montreal in November 2003. An intensive 4 day Jamboree Workshop was organized for Ann Arbor in June 2004, at which numerous work groups pursued cross-laboratory analyses and proposed further work. Investigators were advised to adopt more stringent criteria for high confidence peptide and protein identifications, and a commitment was made to collect raw spectra from the 18 laboratories that had submitted MS/MS or FT-ICR/ MS datasets for independent analyses by three different groups. The datasets were moving targets, as some, but not all, labs submitted expanded or updated analyses, and about 15 laboratories completed "special projects" stimulated by HUPO PPP with a competition for small grants following the Montreal workshop.

The PPP provided participating laboratories with 1.0 mL of reference specimens of serum and plasma by three different methods of anticoagulation for plasma (EDTA, citrate, heparin) from specific donor pools. Investigators utilized their established and emerging technologies for fractionation and analysis of proteins. Investigators were encouraged to "push the limits" of their methods to detect and identify low abundance proteins. Comparisons of findings across laboratories provide a special opportunity for confirmation of protein identifications. Results were submitted to centralized bioinformatics functions at the University of Michigan and the European Bioinformatics Institute to create an integrated data repository from which PPP and other investigators could initiate further analyses and annotations. The approaches and core results have been presented at the US HUPO inaugural meeting in March 2005, the HUPO World Congress in Munich in August 2005, and at other meetings.

Here we present a comprehensive account of the major findings from the pilot phase of the Human Plasma Proteome Project, including the many associated special projects.

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