Science Spotlight

A web-based portal for proteomic assays

The CPTAC Assay Portal is a searchable database of well-characterized targeted proteomic assays.
Image provided by Dr. Henry Rodriguez, Director of the Office of Cancer Clinical Proteomics Research, NCI

Proteins are the workhorses of our cells, carrying out a broad range of biological functions. Remarkably, over a decade since the completion of the human genome sequence, the amounts of the majority of human proteins cannot be measured quantitatively, and the research community is currently reliant on antiquated protein detection methods (e.g. Western blotting, immunohistochemistry) that are semi-quantitative, irreproducible, and often non-specific. The lack of standardized tools for reliably quantifying human proteins not only contributes to the irreproducibility of preclinical research but also renders most of the exciting leads coming from genomics experiments not actionable and renders the human proteome clinically inaccessible. The cost of these antiquated methods is significant, since the research use market for protein-detection antibodies is $2 billion/year, and it is well documented that over half do not work as claimed. This inability to reproducibly quantify proteins in an accurate manner hinders the ability of researchers to look globally at the proteome for novel protein biomarkers or drug targets to understand the basis of human disease.

According to Dr. Amanda Paulovich of the Clinical Research Division, "We are chipping away at this roadblock by building an open-source, biologically-oriented community resource of standardized assays for quantifying proteins." Development of a new web-based portal for validated proteomic assays was led by Dr. Paulovich and collaborating investigators from the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC), and described in the journal Nature Methods.

The CPTAC Assay Portal ( is a public database for well-characterized, targeted proteomic assays. The portal also serves as a public resource for disseminating standard operating protocols, reagents, and assay characterization data. Until now, the information for the hundreds of targeted mass spectrometry-based assays was distributed across published journal articles. Staff scientist Dr. Jeff Whiteaker states that "by incorporating guidelines for assay characterization, the portal takes an important step forward in enabling researchers to assess the performance of each assay. Furthermore, all the data and information for implementing the assays are available for download." 

The overall goal of the portal is to bring together clinicians or biologists with analytical chemists to answer hypothesis-driven questions. The first level of the portal enables investigators to find assays to proteins relevant to their areas of interest by querying specific cellular pathways, protein complexes, or chromosomal locations. The second level of the portal is geared towards mapping assay peptide analytes to protein features of interest, including protein isoforms, domains, polymorphisms, or post-translational modifications. The third level provides the details of the assays to evaluate expected performance, through the program Panorama, an open-source platform for efficient collection and sharing of data created by collaborators at the Department of Genome Sciences at the University of Washington (Sharma et al., 2014). Guidelines are included for the minimal characterization data required for including proteomic assays to the portal. Finally, the fourth level enables the user to preform the assays in their laboratories and a discussion board allows researchers to exchange information for each assay.

The major type of targeted proteomic assay included in the Assay Portal uses multiple reaction monitoring mass spectrometry (MRM-MS). MRM-MS measures proteins using specialized instruments tuned to look for small protein fragments of interest in biological specimens. Proteins are digested with a protease, such as trypsin, to generate smaller peptide fragments. One or more unique peptides are identified for each protein, and used as a surrogate to quantify that protein’s abundance. By focusing the full analytical capacity of the instrument on analytes of interest, MRM-MS increases sensitivity and reproducibility compared to traditional untargeted ("shotgun") proteomic approaches. A previous international study led by the Paulovich Laboratory (Kennedy et al., 2014) demonstrated the feasibility and utility of developing standardized MRM assays to all human proteins to facilitate basic and translational studies by enabling highly multiplexed, precise proteomic measurements to be standardized across laboratories (Whiteaker et al., 2014).

Currently, the CPTAC Assay Portal contains 554 unique MRM assays from the preliminary study on breast cancer (Kennedy et al., 2014), and the CPTAC network will expand this to >1,000 assays over the next few years.  Work is currently underway to open the portal for submissions of well characterized assays from the community, which is expected to further expand content.  The launch of this portal will promote the widespread use of targeted proteomics to address important biological questions on human disease.

As a next step, Dr. Paulovich and her colleagues are working to develop an international public-private partnership to build on the CPTAC Assay Portal architecture and develop a large-scale, standardized, open-source protein assay resource for the community. The goal is to standardize protein quantification and harmonize results across laboratories, improving our ability to validate molecular signatures and the reproducibility of preclinical research, as well as facilitating the regulatory approval of new drugs and diagnostics.

Whiteaker JR, Halusa GN, Hoofnagle AN, Sharma V, MacLean B, Yan P, Wrobel JA, Kennedy J, Mani DR, Zimmerman LJ, Meyer MR, Mesri M, Rodriguez H; Clinical Proteomic Tumor Analysis Consortium (CPTAC), Paulovich AG. (2014) CPTAC Assay Portal: a repository of targeted proteomic assays. Nat Methods 11:703-704.

See also: Kennedy JJ, Abbatiello SE, Kim K, Yan P, Whiteaker JR, Lin C, Kim JS, Zhang Y, Wang X, Ivey RG, Zhao L, Min H, Lee Y, Yu M-H, Yang EG, Lee C, Wang P, Rodriguez H, Kim Y, Carr SA, Paulovich AG. 2014. Demonstrating the feasibility of large-scale development of standardized assays to quantify human proteins. Nat Methods 11, 149–155.

Sharma V, Eckels J, Taylor GK, Shulman NJ, Stergachis AB, Joyner SA, Yan P, Whiteaker JR, Halusa GN, Schilling B, Gibson BW, Colangelo CM, Paulovich AG, Carr SA, Jaffe JD, MacCoss MJ, MacLean B. 2014. Panorama: A Targeted Proteomics Knowledge Base. J Proteome Res Epub ahead of print, doi: 10.1021/pr5006636.