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Demonstrating the feasibility of large-scale development of standardized assays to quantify human proteins


Multiple reaction monitoring (MRM) mass spectrometry has been successfully applied to monitor targeted proteins in biological specimens, raising the possibility that assays could be configured to measure all human proteins. We report the results of a pilot study designed to test the feasibility of a large-scale, international effort for MRM assay generation. We have configured, validated across three laboratories and made publicly available as a resource to the community 645 novel MRM assays representing 319 proteins expressed in human breast cancer. Assays were multiplexed in groups of >150 peptides and deployed to quantify endogenous analytes in a panel of breast cancer–related cell lines. The median assay precision was 5.4%, with high interlaboratory correlation (R2 > 0.96). Peptide measurements in breast cancer cell lines were able to discriminate among molecular subtypes and identify genome-driven changes in the cancer proteome. These results establish the feasibility of a large-scale effort to develop an MRM assay resource.

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Figure 1: Overview of cell-line sample preparation, distribution and MRM analysis.
Figure 2: Analysis of cell lysates shows excellent precision of MRM-based measurements with high correlation and agreement between sites.
Figure 3: Protein and RNA expression data show different genes significantly associated with HER2, ER and basal-luminal23 status.
Figure 4: Distribution of protein expression levels, RNA expression levels and DNA copy numbers of the 12 subtype-enriched genes among genomic and proteomic data sets.
Figure 5: Kaplan-Meier (KM) survival curves of breast cancer patients are stratified by their expression levels of DPYSL2, CLTC or ABAT.


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We are grateful to the Fred Hutchinson Cancer Research Center–University of Washington Breast Specimen Repository and Registry (BSRR) for specimens used in this study. The BSRR is generously supported by the Breast Cancer Relief Foundation, The Foster Foundation and Hutchinson Center funds. All BSRR specimens and data have been obtained in accordance with all applicable human subjects laws and regulations, including any requiring informed consent. The authors thank S. Skates, D. Ransohoff and L. Anderson for helpful discussions. Research reported in this publication was supported in part by the Office of the Director, US National Institutes of Health (NIH OD), and the NCI with funds from the American Recovery and Reinvestment Act of 2009 under grant RC2CA148286. The research was also partially supported by NIH grant U24CA160034 from the NCI Clinical Proteomics Tumor Analysis Consortium Initiative and NIH grant P50CA138293 from the NCI Specialized Programs of Research Excellence (SPORE). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. This research was also partially supported by the Proteogenomic Research Program through the National Research Foundation of Korea, funded by the Korea government (MSIP), and correspondence to the Seoul site should be addressed to Y.K. (

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Authors and Affiliations



J.J.K., S.E.A., K.K., J.R.W., P.W., Y.K., S.A.C. and A.G.P. conceived of and designed the experiments. J.J.K., S.E.A., K.K., J.S.K., R.G.I., L.Z., Y.L. and H.M. performed the experiments. J.J.K., S.E.A., K.K., P.Y., C. Lin, Y.Z. and X.W. analyzed the data. M.-H.Y., E.G.Y., C. Lee, H.R., Y.K., S.A.C. and A.G.P. contributed reagents, materials and/or analysis tools. J.J.K., J.R.W. and A.G.P. wrote the paper.

Corresponding author

Correspondence to Amanda G Paulovich.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Protocol and Supplementary Results (PDF 9126 kb)

Supplementary Table 1

Summary of the breast cancer cell lines and tumor tissue samples assayed in the study. (XLSX 14 kb)

Supplementary Table 2

Summary of MRM assay parameters for the selected peptide and protein targets in the study. (XLSX 284 kb)

Supplementary Table 3

MRM assay characteristics and figures of merit. (XLSX 599 kb)

Supplementary Table 4

Endogenous levels measured by MRM in 30 cell lines. (XLSX 764 kb)

Supplementary Table 5

Transitions included in the MRM measurement of endogenous levels of for the selected peptide and protein targets in the study. (XLSX 128 kb)

Supplementary Table 6

Precision of the endogenous levels measurements by MRM in 30 cell lines. (XLSX 266 kb)

Supplementary Table 7

Proteins with observed differential expression in ER+ vs. ER-, HER2+ vs. HER2- and basal vs. luminal subtypes. (XLSX 85 kb)

Supplementary Table 8

Integrative analysis results for evaluating the concordance across DNA copy numbers, gene expression and protein expression. (XLSX 35 kb)

Supplementary Table 9

Summary of Cox survival analysis for DPYSL2, CLTC, PRDX3, ALDOA ANXA1, ABAT, GALK1, PLOD3 and CDKN2A based on NKI and LOI data sets. (XLSX 13 kb)

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Kennedy, J., Abbatiello, S., Kim, K. et al. Demonstrating the feasibility of large-scale development of standardized assays to quantify human proteins. Nat Methods 11, 149–155 (2014).

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