Abstract
Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low μg/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma.
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Change history
09 September 2009
In the version of this article initially published, the following acknowledgment was inadvertently left out: “The UCSF CPTAC team gratefully acknowledges the support of the Canary Foundation for providing funds to purchase a 4000 QTRAP mass spectrometer.” The acknowlegment has been added to the HTML and PDF versions of the article.
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Acknowledgements
This work was supported by grants from the National Cancer Institute (NCI) (U24 CA126476, U24 126477, U24 126480, U24 CA126485, and U24 126479), part of NCI Clinical Proteomic Technologies for Cancer initiative. A component of this initiative is the Clinical Proteomic Technology Assessment for Cancer (CPTAC) Network and teams, which include the Broad Institute of MIT 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). A full listing of the CPTAC Team Network can be found at http://proteomics.cancer.gov/programs/CPTAC/networkmembership. The UCSF CPTAC team gratefully acknowledges the support of the Canary Foundation for providing funds to purchase a 4000 QTRAP mass spectrometer.
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The CPTAC Network contributed collectively to this study. The following CPTAC Network investigators contributed significant intellectual contributions to work described in this paper.
S.E.A., T.A., N.L.A., D.M.B, S.C.H., A.-J.L.H., H.K., D.R., B.S., S.J.S., L.J.Z. and S.A.C. contributed to study design and SOP development. D.M.B and N.G.D. prepared and shipped samples. S.E.A., T.A., S.A., H.L.C., J.M.H., A.J., E.B.J., H.K., D.S., T.J.T., J.R.W., A.W., S.W., L.Z., and L.J.Z. contributed to generation of data. M.P.C., J.L., D.R.M., R.K.N., S.J.S., T.C.P., P.A.R., C.H.S., D.L.T., A.M.V., and L.J.V.-M. contributed to bioinformatics and statistical analysis. S.E.A, T.A., H.K., D.R.M., S.J.S. and L.J.Z. centrally reviewed data. S.E.A., T.A., N.L.A., S.A.C., S.J.F., S.C.H., A.-J.L.H., H.K., D.R.M, B.S., S.J.S., and L.J.Z. wrote and prepared the manuscript. R.K.B., C.B., C.H.B., S.A.C., S.J.F., B.W.G., T.H., C.R.K., D.C.L., M.M., T.A.N., A.G.P., H.R., F.E.R., P.T., and M.W. contributed to experimental design. S.C.H. chaired the CPTAC Experimental Design and Statistics Verification Studies Working Group that designed interlaboratory studies and generated data.
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Addona, T., Abbatiello, S., Schilling, B. et al. Multi-site assessment of the precision and reproducibility of multiple reaction monitoring–based measurements of proteins in plasma. Nat Biotechnol 27, 633–641 (2009). https://doi.org/10.1038/nbt.1546
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DOI: https://doi.org/10.1038/nbt.1546
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