Partners in Proteomics

---

By Elizabeth Cooper

The Human Proteome Organization (HUPO), with its eight International Scientific Research Initiatives, is at the forefront of the burgeoning field of proteomics. HUPO’s New Technology & Resources Committee and Standards Committee work closely with its seven-member Industry Advisory Council and its Education and Training Committee to ensure technology transfer and a high standard of scientific output.

HUPO’s involvement with proteomic data standards through the Proteomic Standards Initiative, and biomarker development through the Biomarker Initiative and Plasma Proteome Project, has raised the bar with respect to the quality and applicability of proteomic research. In addition, HUPO scientists are developing state-of-the-art equipment and tools to improve current mass spectrometry (MS) technological issues such as noise problems when complex protein mixtures are analyzed.

Standardization Efforts
The driving forces for the development of timely, harmonized and globally available proteomic standards are fundamentally economic, medical and scientific. In order to develop new therapeutic and diagnostic tools, large pharmaceutical companies need to know which proteins are involved in a disease process. This requires good baseline plasma and serum proteomics data for normal human populations.

HUPO, along with the U.S. National Institute of Standards and Technology, the U.S. National Institutes of Health, the Canadian Institutes of Health Research (Ottawa, ON), Genome Canada (Ottawa, ON), the European Commission — as well as industry partners such as Invitrogen Corp. (Carlsbad, CA), NonLinear Dynamics Ltd. (Newcastle upon Tyne, U.K.) and GenoLogics LifeScience Software Inc. (Victoria, BC) — has been involved in several proteomics-related standardization efforts that range from data-analysis issues to the development and implementation of protein test standards. All HUPO scientific initiatives adhere to the data standards being developed by HUPO’s Proteomic Standards Initiative, based at the European Bioinformatics Institute in the U.K. This Initiative is in the process of co-ordinating its efforts with the Institute for Systems Biology (Seattle, WA), another major bioinformatics-related consortium.

HUPO’s Plasma Proteome Project, which is in its pilot phase, includes labs from 14 countries and is one of several HUPO and non-HUPO research efforts that are using proteomic reference specimens. This project has generated extensive inter-laboratory data for protein identifications in serum and in EDTA-, heparin-, and citrate-anticoagulated human plasma. As part of this project, four independent laboratories performed quantitative immunoassays on more than 200 protein analytes in common reference specimens. The Plasma Proteome Project published its results in 28 papers in Proteomics in August 2005 and additional informatics methods in Nature Biotechnology in March 2006. EDTA-plasma was recommended as the preferred blood specimen for biomarker studies. Quantitative proteomic methods used by this project and other proteomic standardization efforts must be able to deal with complex specimens. Techniques used include isotopic labelling methods such as ICAT, SILAC, and iTRAQ.
HUPO’s Human Antibody Initiative, based in Sweden, has been set up to allow the systematic exploration of the human proteome with affinity (antibody) proteomics, combining high-throughput generation of affinity-purified (mono-specific) antibodies with protein profiling using tissue arrays (Figure 1). The resultant Human Protein Atlas provides free public access to more than 800 validated proteins. A standardization effort is underway to utilize the proteins in this database for mapping the human genome and proteome.

Biomarker Activities – Cardiovascular
Modern medical practice is based largely upon defining disease via (i) records of patient history; (ii) data supporting diagnosis and prognosis of new or ongoing pathological phenotypes; and (iii) various clinical laboratory parameters that enable selection and guidance of effective treatments.

In the past decade, the use of sophisticated markers to facilitate health care in cardiovascular disease has primarily focused on their applications to prognosis/diagnosis. In contrast, protein markers indicative of patient history, or protein markers facilitating the selection and guidance of therapy, are largely absent.

The Cardiovascular Initiative of HUPO Biomarkers aims to address the above issues. This initiative is employing proteomic technologies that allow characterization of functional subproteomes coupled with carefully constructed clinical databases and sophisticated analytic techniques, to bridge our nascent appreciation of biological complexity to our ultimate goal of understanding and treating disease. This effort includes a thorough understanding of the temporal profile of protein abnormalities associated with major cardiovascular diseases, as well as the delineation of functional proteomic changes in cardiovascular tissues that maybe used for either prognostic or therapeutic guidance.

New Technologies Being Developed – A Microfluidic Robot
Although MS — the method of choice for quantitative, large scale protein profiling — has shown tremendous progress, it is an inherently sequential approach and works better with purified samples rather than with the complex specimens usually available for diagnosis. Biomarkers identified using such methods often still need to be validated using classical enzyme-linked immunosorbent assays (ELISAs). Protein microarrays also have a complementary value to MS in that they allow the examination of lower abundance proteins, membrane proteins and proteins with extreme pI values. Protein microarrays represent a parallel approach; as with other methods, they must overcome nonspecific binding of proteins present in high abundance.

Researchers, based at McGill University (Montreal, QC), are proposing an antibody-based approach that combines a microarray format, an assay architecture that emulates ELISA, and microfluidic flow control. The benefit of this approach is that the noise on each spot can be decoupled from the size of the array, as in ELISA, and thus large-scale proteomics can be transformed into a tractable technological problem, instead of an intractable biological one. This team is building a micofluidic proteomics platform with high specificity amenable to large-scale proteomics of human bodily fluids, compatible with the demands of clinical diagnostics.

The outcome of this project (Figure 2) will be a workable prototype for large scale Ab-based proteomics and will constitute a credible proof-of-concept for a compartmentalized protein microarray, or equivalently, for an ultraminiaturized and parallelized immunosorbent assay platform that has no intrinsic upper limit to the application to all Abs specific to each expressed gene of the human genome.
The associated large-scale, antibody-based proteomic platform can also be a universal protein signature profiling system because of its sensitivity, throughput, and overall competitive cost. Such a platform will therefore find application in the early diagnosis of cancer, in the prognosis of cancer progression, and in monitoring the treatment of cancer and other diseases. Already the researchers involved in this project have developed a new class of autonomous microfluidic capillary systems (Figure 3) and a miniaturized, multiplexed test covering conventional and putative cardiac disease markers.

This newly designed and built proteomics platform for protein profiling is based on microfluidic technologies capable of supporting a sandwich immunoassay format with co-localization of cognate pairs of Abs at the scale of standard microarrays.
This platform is expected also to provide new insight on the protein signature profiles of breast cancers from the detailed analysis of protein profiles in tissue and serum or plasma.

Conclusion
The complementary relationship between HUPO and various industrial partners will reduce the time taken from basic research to diagnostic tools and therapeutics. HUPO’s interest in continuing to involve more laboratories — academic and industrial — will undoubtedly improve the long-term outlook for this field.