First-of-its-kind study fills key proteomic gap

Image courtesy of National Cancer Institute

Harbingers of disease come in several forms, but there is increasing interest in molecular signposts, particularly proteins, that can act as early warning signals for doctors and their patients. But before such protein “biomarkers” can be deployed in the clinic, they have to be identified in the laboratory and then repeatedly tested in hundreds, preferably thousands, of patient samples to assure that they are effective indicators of disease. It is a long and arduous path from the bench to the clinic, one that is made even more challenging by several key technological gaps in the large-scale analysis of proteins (known as “proteomics”).

Now, a first-of-its-kind study appearing in the June 28 advance online edition of Nature Biotechnology helps bridge those gaps by offering a method to detect and quantify multiple protein biomarkers in blood samples. Most important, this method can be widely reproduced in different laboratories using varied equipment, a feat once considered largely impossible. The work was led by five research institutes, including the Broad Institute, as part of the National Cancer Institute’s Clinical Proteomic Technology Assessment for Cancer (CPTAC).

“This is a first step in the long march to move protein biomarkers from the laboratory into the clinic,” said senior author Steven Carr, a CPTAC investigator and director of the Broad Institute’s Proteomics Platform. “In addition to a critical analysis of proteomic technology, we’ve also created an important community resource. Our reagents, protocols and data are available to any researcher who wants to use them.”

At the heart of the Nature Biotechnology study is a three-part analysis of a standard sample of seven mixed proteins. CPTAC researchers independently measured the relative levels of these proteins using a method that can simultaneously detect multiple proteins, known as multiple reaction monitoring coupled with stable isotope dilution mass spectrometry or SID-MRM-MS, and then shared their results. “We designed our study to introduce variability incrementally, so we could systematically tease apart sources of variation that operate not only within a laboratory but between laboratories,” said co-author Susan Abbatiello, a research scientist in the Broad’s Proteomics Platform.

Not only was this work carried out in different laboratories by different researchers, but often using different types of instruments, which introduced yet another source of variability.

The researchers also shared and analyzed their results in a coordinated way, ultimately making the data and analytical techniques available to the proteomics community. “The statistical analyses were another huge challenge, to track and process data from all of the participating labs,” said co-author D.R. Mani, a computational biologist at the Broad Institute.

Yet all of their efforts seem to have paid off. Carr and his CPTAC colleagues demonstrated that the SID-MRM-MS approach yields consistent results, within a single laboratory as well as across multiple sites. This method of testing patient samples is highly sensitive, meaning it can detect proteins that are present in relatively low concentrations. This is critical since most clinically relevant protein biomarkers are present at low levels.  Future efforts will focus on further enhancing the method’s sensitivity.  

In addition, the researchers showed that their method can be successfully applied to samples derived from blood — a significant step because blood is among the most convenient body fluids to analyze in the clinic. But blood is also a highly complex mixture, involving hundreds of thousands of different proteins in a vast range of concentrations. That mishmash is notoriously difficult for protein scientists to analyze.

To further disperse their hard-won knowledge, Carr and his colleagues are following up their Nature Biotechnology paper with a two-day course to train laboratory staff who work with proteomic equipment. Given the multiple steps involved in proteomic analyses, they discovered that such tactical discussions were a critical part of the trouble-shooting process. The course will initially be geared toward researchers in the CPTAC network, but Carr says it may be available in the future to the broader scientific community.

The CPTAC researchers face an even steeper challenge in the next phase of work: analyzing samples with a 10-fold increase in biomarker targets and across a wider network of laboratories. But that kind of proteomic “heavy-lifting” is urgently needed before protein biomarkers can be incorporated into routine clinical care.

The full list of institutes participating in the CPTAC study includes the Broad Institute of the Massachusetts Institute of Technology and Harvard (with the Fred Hutchinson Cancer Research Center, Massachusetts General Hospital, the University of North Carolina at Chapel Hill, the University of Victoria and the Plasma Proteome Institute), Memorial Sloan-Kettering Cancer Center (with the Skirball Institute at New York University), Purdue University (with Monarch Life Sciences, Indiana University, Indiana University-Purdue University Indianapolis and the Hoosier Oncology Group ), University of California, San Francisco (with the Buck Institute for Age Research, Lawrence Berkeley National Laboratory, the University of British Columbia and the University of Texas M.D. Anderson Cancer Center), and Vanderbilt University School of Medicine (with the University of Texas M.D. Anderson Cancer Center, the University of Washington and the University of Arizona).