Targeted mass-spectrometry-based assays enable multiplex quantification of receptor tyrosine kinase, MAP kinase, and AKT signaling
Summary: A primary goal of the US National Cancer Institute's Ras initiative at the Frederick National Laboratory for Cancer Research is to develop methods to quantify RAS signaling to facilitate development of novel cancer therapeutics. We use targeted proteomics technologies to develop a comm...
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Format: | Article |
Language: | English |
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Elsevier
2021-07-01
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Series: | Cell Reports: Methods |
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Online Access: | http://www.sciencedirect.com/science/article/pii/S2667237521000151 |
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doaj-2467da2d3b9c429ca7d2d7f7a1e3f48e |
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record_format |
Article |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Jeffrey R. Whiteaker Kanika Sharma Melissa A. Hoffman Eric Kuhn Lei Zhao Alexandra R. Cocco Regine M. Schoenherr Jacob J. Kennedy Ulianna Voytovich Chenwei Lin Bin Fang Kiah Bowers Gordon Whiteley Simona Colantonio William Bocik Rhonda Roberts Tara Hiltke Emily Boja Henry Rodriguez Frank McCormick Matthew Holderfield Steven A. Carr John M. Koomen Amanda G. Paulovich |
spellingShingle |
Jeffrey R. Whiteaker Kanika Sharma Melissa A. Hoffman Eric Kuhn Lei Zhao Alexandra R. Cocco Regine M. Schoenherr Jacob J. Kennedy Ulianna Voytovich Chenwei Lin Bin Fang Kiah Bowers Gordon Whiteley Simona Colantonio William Bocik Rhonda Roberts Tara Hiltke Emily Boja Henry Rodriguez Frank McCormick Matthew Holderfield Steven A. Carr John M. Koomen Amanda G. Paulovich Targeted mass-spectrometry-based assays enable multiplex quantification of receptor tyrosine kinase, MAP kinase, and AKT signaling Cell Reports: Methods cancer signaling immuno-MRM assay resource quantitative proteomics pharmacodynamics targeted therapy |
author_facet |
Jeffrey R. Whiteaker Kanika Sharma Melissa A. Hoffman Eric Kuhn Lei Zhao Alexandra R. Cocco Regine M. Schoenherr Jacob J. Kennedy Ulianna Voytovich Chenwei Lin Bin Fang Kiah Bowers Gordon Whiteley Simona Colantonio William Bocik Rhonda Roberts Tara Hiltke Emily Boja Henry Rodriguez Frank McCormick Matthew Holderfield Steven A. Carr John M. Koomen Amanda G. Paulovich |
author_sort |
Jeffrey R. Whiteaker |
title |
Targeted mass-spectrometry-based assays enable multiplex quantification of receptor tyrosine kinase, MAP kinase, and AKT signaling |
title_short |
Targeted mass-spectrometry-based assays enable multiplex quantification of receptor tyrosine kinase, MAP kinase, and AKT signaling |
title_full |
Targeted mass-spectrometry-based assays enable multiplex quantification of receptor tyrosine kinase, MAP kinase, and AKT signaling |
title_fullStr |
Targeted mass-spectrometry-based assays enable multiplex quantification of receptor tyrosine kinase, MAP kinase, and AKT signaling |
title_full_unstemmed |
Targeted mass-spectrometry-based assays enable multiplex quantification of receptor tyrosine kinase, MAP kinase, and AKT signaling |
title_sort |
targeted mass-spectrometry-based assays enable multiplex quantification of receptor tyrosine kinase, map kinase, and akt signaling |
publisher |
Elsevier |
series |
Cell Reports: Methods |
issn |
2667-2375 |
publishDate |
2021-07-01 |
description |
Summary: A primary goal of the US National Cancer Institute's Ras initiative at the Frederick National Laboratory for Cancer Research is to develop methods to quantify RAS signaling to facilitate development of novel cancer therapeutics. We use targeted proteomics technologies to develop a community resource consisting of 256 validated multiple reaction monitoring (MRM)-based, multiplexed assays for quantifying protein expression and phosphorylation through the receptor tyrosine kinase, MAPK, and AKT signaling networks. As proof of concept, we quantify the response of melanoma (A375 and SK-MEL-2) and colorectal cancer (HCT-116 and HT-29) cell lines to BRAF inhibition by PLX4720. These assays replace over 60 western blots with quantitative mass-spectrometry-based assays of high molecular specificity and quantitative precision, showing the value of these methods for pharmacodynamic measurements and mechanism-of-action studies. Methods, fit-for-purpose validation, and results are publicly available as a resource for the community at assays.cancer.gov. Motivation: A lack of quantitative, multiplexable assays for phosphosignaling limits comprehensive investigation of aberrant signaling in cancer and evaluation of novel treatments. To alleviate this limitation, we sought to develop assays by using targeted mass spectrometry for quantifying protein expression and phosphorylation through the receptor tyrosine kinase, MAPK, and AKT signaling networks. The resulting assays provide a resource for replacing over 60 western blots in examining cancer signaling and tumor biology with high molecular specificity and quantitative rigor. |
topic |
cancer signaling immuno-MRM assay resource quantitative proteomics pharmacodynamics targeted therapy |
url |
http://www.sciencedirect.com/science/article/pii/S2667237521000151 |
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doaj-2467da2d3b9c429ca7d2d7f7a1e3f48e2021-09-03T04:48:44ZengElsevierCell Reports: Methods2667-23752021-07-0113100015Targeted mass-spectrometry-based assays enable multiplex quantification of receptor tyrosine kinase, MAP kinase, and AKT signalingJeffrey R. Whiteaker0Kanika Sharma1Melissa A. Hoffman2Eric Kuhn3Lei Zhao4Alexandra R. Cocco5Regine M. Schoenherr6Jacob J. Kennedy7Ulianna Voytovich8Chenwei Lin9Bin Fang10Kiah Bowers11Gordon Whiteley12Simona Colantonio13William Bocik14Rhonda Roberts15Tara Hiltke16Emily Boja17Henry Rodriguez18Frank McCormick19Matthew Holderfield20Steven A. Carr21John M. Koomen22Amanda G. Paulovich23Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USANCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21701, USAProteomics and Metabolomics Core, Department of Molecular Oncology, and Department of Tumor Biology, Moffitt Cancer Center & Research Institute, Tampa, FL 33612, USABroad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA 02142, USAClinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USAGillings School of Global Public Health, Kenan-Flagler Business School, University of North Carolina, Chapel Hill, NC 27599, USAClinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USAClinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USAClinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USAClinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USAProteomics and Metabolomics Core, Department of Molecular Oncology, and Department of Tumor Biology, Moffitt Cancer Center & Research Institute, Tampa, FL 33612, USAProteomics and Metabolomics Core, Department of Molecular Oncology, and Department of Tumor Biology, Moffitt Cancer Center & Research Institute, Tampa, FL 33612, USAAntibody Characterization Laboratory, Leidos Biochemical Research Inc, Frederick National Laboratory for Cancer Research ATRF, Frederick, MD 21701, USAAntibody Characterization Laboratory, Leidos Biochemical Research Inc, Frederick National Laboratory for Cancer Research ATRF, Frederick, MD 21701, USAAntibody Characterization Laboratory, Leidos Biochemical Research Inc, Frederick National Laboratory for Cancer Research ATRF, Frederick, MD 21701, USAAntibody Characterization Laboratory, Leidos Biochemical Research Inc, Frederick National Laboratory for Cancer Research ATRF, Frederick, MD 21701, USAOffice of Cancer Clinical Proteomics Research, National Cancer Institute, Bethesda, MD 20892, USAOffice of Cancer Clinical Proteomics Research, National Cancer Institute, Bethesda, MD 20892, USAOffice of Cancer Clinical Proteomics Research, National Cancer Institute, Bethesda, MD 20892, USANCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21701, USA; Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA 94158, USANCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21701, USA; Department of Biology, Revolution Medicines, Inc., Redwood City, CA 94063, USABroad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA 02142, USAProteomics and Metabolomics Core, Department of Molecular Oncology, and Department of Tumor Biology, Moffitt Cancer Center & Research Institute, Tampa, FL 33612, USA; Corresponding authorClinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; Corresponding authorSummary: A primary goal of the US National Cancer Institute's Ras initiative at the Frederick National Laboratory for Cancer Research is to develop methods to quantify RAS signaling to facilitate development of novel cancer therapeutics. We use targeted proteomics technologies to develop a community resource consisting of 256 validated multiple reaction monitoring (MRM)-based, multiplexed assays for quantifying protein expression and phosphorylation through the receptor tyrosine kinase, MAPK, and AKT signaling networks. As proof of concept, we quantify the response of melanoma (A375 and SK-MEL-2) and colorectal cancer (HCT-116 and HT-29) cell lines to BRAF inhibition by PLX4720. These assays replace over 60 western blots with quantitative mass-spectrometry-based assays of high molecular specificity and quantitative precision, showing the value of these methods for pharmacodynamic measurements and mechanism-of-action studies. Methods, fit-for-purpose validation, and results are publicly available as a resource for the community at assays.cancer.gov. Motivation: A lack of quantitative, multiplexable assays for phosphosignaling limits comprehensive investigation of aberrant signaling in cancer and evaluation of novel treatments. To alleviate this limitation, we sought to develop assays by using targeted mass spectrometry for quantifying protein expression and phosphorylation through the receptor tyrosine kinase, MAPK, and AKT signaling networks. The resulting assays provide a resource for replacing over 60 western blots in examining cancer signaling and tumor biology with high molecular specificity and quantitative rigor.http://www.sciencedirect.com/science/article/pii/S2667237521000151cancer signalingimmuno-MRMassay resourcequantitative proteomicspharmacodynamicstargeted therapy |