This story originally ran on July 8.

By Tony Fong

Addressing one of the most formidable bottlenecks in proteomics, a team of researchers put together by the National Cancer Institute three years ago has demonstrated for the first time that a relatively new verification method is reproducible across different laboratories.

Individual labs have shown that multiple reaction monitoring coupled with isotope dilution mass spectrometry, or SID-MRM-MS, can be used to quantify candidate protein biomarkers in plasma, but until now no one had proven the interlaboratory reproducibility of the method or the transferability of the assays across different labs.

But using "common materials" and standardized protocols, a multisite collaboration has shown that different labs can come up with the same data for the same experiment. The work, described in an article published June 28 in the online edition of Nature Biotechnology, also shows sensitivity for the method to the low microgram per milliliter protein concentrations in unfractionated plasma.

The work is the first of a series of studies to come out from the working groups of NCI's Clinical Proteomic Technology Assessment for Cancer. In 2006 under that program, the NCI awarded five teams $35.5 million over five years to evaluate proteomics technologies with applications to cancer research [see PM 09/28/06].

In addition to the current study, other interlaboratory studies related to discovery proteomics are in review, Steven Carr, senior author on the study and director of the Proteomics Platform at the Broad Institute, told ProteoMonitor last week. And the working group responsible for last week's publication is preparing a "second major study" focusing on "substantially improving sensitivity as well as increasing the plex-level of [approximately] 10-plex to 100-plex," Carr said.

The current study is focused on a new application for SID-MRM-MS, a method that has been in development and use for several years now.

"The primary importance of the work … has to deal with the fact that there is a critical need for a technology that will allow us to move so-called discoveries coming out of the -omics methodologies … forward," Carr said. "The issue is that discovery experiments do not lead to biomarkers in clinical proteomics. They lead to hypotheses or candidates that need to be further credentialed."

Discovery proteomics often results in a list of tens or even hundreds of potential biomarkers, but because analyzing each biomarker can take up to several weeks and the sample numbers are low, the false discovery rates tend to run high, Carr said. That is not necessarily because of technical variability — though he added that that is something that is still not completely understood — but rather is the consequence of biological variability in the samples.

The task is to identify the changes due to pathology, but to do that, researchers need a step to help them bridge the gulf between discovery and "any possibility of clinical utility," Carr said.

Initial potential biomarkers have been typically verified through sensitive and specific high-throughput immunoassays, but such technology has been limited by the availability of well-characterized antibodies, a well-known problem in protein research. Developing high-quality immunoassays also is costly and time-consuming.

As a result, there is a need for more "straightforward quantitative approaches, exploiting the sensitivity and molecular specificity of mass spectrometry," Carr and his co-authors wrote.

In their study, they cite work suggesting SID-MRM-MS as a promising strategy for the direct quantitation of proteins in cell lysates and human plasma and serum. Several members of the CPTAC team, including Carr as well as Mandy Paulovich at the Fred Hutchinson Cancer Research Center, also were already developing new MRM-MS workflows, though other labs participating in the study had little or no experience with the MRM workflow.

At the Broad, Carr's lab has shown that such methods, combined with protein- and peptide-enrichment strategies, are able to "hit target values for limits of quantitation that are in the very bottom of the nanogram per milliliter range for proteins in blood," where many biomarkers of clinical utility reside, he said.

While such work suggests SID-MRM-MS may be suitable for the kind of work called for in biomarker verification — the rapid screening of large numbers of candidate protein biomarkers in large-scale patient samples — the adoption of the method has been tepid because of questions about its interlaboratory reproducibility.