We propose to assess the robustness of a high-throughput mass spectrometric technology platform for quantitative measurement of multiple candidate biomarker proteins in complex samples such as human plasma. Specific assays will be developed for 300 candidate biomarker proteins drawn from published literature, pathway analysis, microarray, and proteomics discovery efforts. These assays will be used to measure biomarker levels in 200 selected cancer and 200 control plasma samples at three different laboratory sites using two variant MS platforms. The results will characterize the quantitative reproducibility of the assay methodology within- and between-laboratories, and over time. A series of standardized reagents will be developed allowing the assays to be implemented in other laboratories using similar standardized instrumentation. Our measurement platform makes use of well-established quantitative MS techniques used in the routine measurement of small molecules - so-called """"""""multiple reaction monitoring"""""""" (or MRM) assays and stable isotope labeled internal standards for quantitation, here in the form of synthetic, labeled peptides. By measuring both the labeled and unlabeled (sample-derived) peptides by MS, the method provides a quantitative measure of the relative amounts of the signature peptide and therefore the protein that is derived from. In order to access lower abundance biomarkers (those in the lower 5 orders of magnitude of the overall 10 order plasma abundance scale) we will employ specific enrichment techniques using immobilized anti-peptide antibodies. After extensive optimization and characterization, our assays will be deployed on clinical plasma samples from breast cancer cases and controls. Key outcomes of this project will be to 1) demonstrate that we can make sensitive and specific assays quickly and inexpensively; 2) that the assays can be highly multiplexed, greatly reducing the cost-per-analyte; and 3) that the protocols and technology can be standardized and distributed. Several members of our CRTAC team are currently involved in proteomic consortia aimed at MS-based discovery of candidate breast cancer biomarkers in human samples and mouse models. These efforts are generating robust datasets that will also be leveraged by this CPTAC team to inform selection of candidates for assay development and ultimate validation.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Resource-Related Research Projects--Cooperative Agreements (U24)
Project #
3U24CA126476-01S1
Application #
7495764
Study Section
Special Emphasis Panel (ZCA1-SRRB-C (O1))
Program Officer
Rodriguez, Henry
Project Start
2006-09-28
Project End
2011-08-31
Budget Start
2006-09-28
Budget End
2007-08-31
Support Year
1
Fiscal Year
2007
Total Cost
$15,000
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Type
Organized Research Units
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02139
Abbatiello, Susan E; Schilling, Birgit; Mani, D R et al. (2015) Large-Scale Interlaboratory Study to Develop, Analytically Validate and Apply Highly Multiplexed, Quantitative Peptide Assays to Measure Cancer-Relevant Proteins in Plasma. Mol Cell Proteomics 14:2357-74
Feng, Xingdong; Sedransk, Nell; Xia, Jessie Q (2014) Calibration using constrained smoothing with applications to mass spectrometry data. Biometrics 70:398-408
Abbatiello, Susan E; Mani, D R; Schilling, Birgit et al. (2013) Design, implementation and multisite evaluation of a system suitability protocol for the quantitative assessment of instrument performance in liquid chromatography-multiple reaction monitoring-MS (LC-MRM-MS). Mol Cell Proteomics 12:2623-39
Whiteaker, Jeffrey R; Zhao, Lei; Lin, Chenwei et al. (2012) Sequential multiplexed analyte quantification using peptide immunoaffinity enrichment coupled to mass spectrometry. Mol Cell Proteomics 11:M111.015347
Guthals, Adrian; Clauser, Karl R; Bandeira, Nuno (2012) Shotgun protein sequencing with meta-contig assembly. Mol Cell Proteomics 11:1084-96
Chalkley, Robert J; Clauser, Karl R (2012) Modification site localization scoring: strategies and performance. Mol Cell Proteomics 11:3-14
Kuhn, Eric; Whiteaker, Jeffrey R; Mani, D R et al. (2012) Interlaboratory evaluation of automated, multiplexed peptide immunoaffinity enrichment coupled to multiple reaction monitoring mass spectrometry for quantifying proteins in plasma. Mol Cell Proteomics 11:M111.013854
Anderson, N Leigh; Razavi, Morteza; Pearson, Terry W et al. (2012) Precision of heavy-light peptide ratios measured by maldi-tof mass spectrometry. J Proteome Res 11:1868-78
Mani, D R; Abbatiello, Susan E; Carr, Steven A (2012) Statistical characterization of multiple-reaction monitoring mass spectrometry (MRM-MS) assays for quantitative proteomics. BMC Bioinformatics 13 Suppl 16:S9
Schoenherr, Regine M; Whiteaker, Jeffrey R; Zhao, Lei et al. (2012) Multiplexed quantification of estrogen receptor and HER2/Neu in tissue and cell lysates by peptide immunoaffinity enrichment mass spectrometry. Proteomics 12:1253-60

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