By Tony Fong

A wide-ranging team of researchers will use a $4.8 million ARRA grant to test the feasibility of measuring all proteins in humans.

The project, which will be funded for two years, will act as a "precursor" study to determine whether the Human Proteome Detection and Quantitation initiative, or hPDQ, can be done.

Participants in the first phase include researchers from the Broad Institute, the Fred Hutchinson Cancer Research Center, the company Plasma Proteome, and others.

First proposed in January in an article published in Molecular and Cellular Proteomics, hPDQ would create a library of anti-peptide antibodies to measure collections of proteins with high sensitivity and specificity [See PM 01/22/09]. The project would serve as a "near-term tactical approach" for providing a more basic map of the human proteome than the Human Proteome Organization's Human Proteome Project.

The current project will determine the feasibility of such a project by testing what it would take to build multiplexed mass spectrometry-based assays, using a multiple-reaction monitoring strategy, to cover the entire human proteome.

Because making and charactering antibodies for such assays are both time-intensive and costly, "it makes sense to first … characterize at the peptide level what assays work really well — what peptides work really well — and then subsequently go in and make antibodies in subsequent phases to those peptides to improve the sensitivity and throughput of the assays," Amanda Paulovich, a co-PI on the project, told ProteoMonitor this week. She is an associate member in the Clinical Research Division at the Fred Hutchinson Cancer Research Center specializing in genetics and oncology.

The project was recently identified by ProteoMonitor as the proteomics-directed research that received the highest stimulus funding in fiscal year 2009 [See PM 10/16/09].

Steven Carr, director of the Proteomics Platform at the Broad Institute, is the other co-PI, and Leigh Anderson, founder and CEO of the Plasma Proteome Institute, is senior advisor on the project.

"So this is the precursor stage where we're focused entirely on the MRM, defining what the best proteotypic peptides are, what the parameters are, and doing exploratory studies and cell lysates to see how well the assays behave," Paulovich added. "And then in a subsequent phase beyond this two-year early phase, we would go on to generate antibodies to improve sensitivity and throughput."

The fact that existing assays can measure only "very few human proteins" currently has been a "major impediment to research," she said. "So the idea here is that if we can achieve the ultimate goal of building a highly specific, sensitive, [and] multiplexible mass spec-based assay for every human protein, that would greatly facilitate not only basic biomedical research but also applied research such as biomarker studies."

Carr emphasized that though the project has a strong clinical angle, the benefits would have a much broader reach for the whole proteomics community.

"It's really about quantitative biology," he said. "While the project is addressing what appears to be a clinical need, it really has extensibility to any biological question you wish to answer in whatever context — tissue, cell line, biofluid — you want to address."

To those ends, Paulovich, Carr, and their colleagues will build assays to proteins detected in frozen breast cancer surgical specimens, then configure and fully characterize them in breast cancer cell lines. The goal is to configure a total of 400 novel assays targeting 400 peptides from 200 proteins.

They will then evaluate the assays in 51 breast cancer cell lines that had been previously characterized from a genomic perspective by Joe Gray's laboratory at the University of California, San Francisco.

Paulovich, Carr, and colleagues chose these particular proteins because they are expressed in human breast cancer and are detectable by mass spectrometry. Just as importantly, they may have some clinical relevance.

They will be evaluating the clinical relevance by mining Matthias Uhlen's Human Protein Atlas to look for proteins that are expressed in breast cancers but not in normal breast epithelium. "Those would be higher priority targets for us," she said.