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Multiplexed Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry Quantification of Cancer Signaling Proteins

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Proteomics for Drug Discovery

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1647))

Abstract

Quantitative evaluation of protein expression across multiple cancer-related signaling pathways (e.g., Wnt/β-catenin, TGF-β, receptor tyrosine kinases (RTK), MAP kinases, NF-κB, and apoptosis) in tumor tissues may enable the development of a molecular profile for each individual tumor that can aid in the selection of appropriate targeted cancer therapies. Here, we describe the development of a broadly applicable protocol to develop and implement quantitative mass spectrometry assays using cell line models and frozen tissue specimens from colon cancer patients. Cell lines are used to develop peptide-based assays for protein quantification, which are incorporated into a method based on SDS-PAGE protein fractionation, in-gel digestion, and liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM/MS). This analytical platform is then applied to frozen tumor tissues. This protocol can be broadly applied to the study of human disease using multiplexed LC-MRM assays.

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Acknowledgments

This work was supported in part by the following core facilities at Moffitt Cancer Center: Tissue Core, Analytic Microscopy, Proteomics, and Biostatistics; funding provided in part by the Cancer Center Support Grant, P30-CA076292, from the National Cancer Institute. Proteomics instruments are supported by funding from the Moffitt Foundation and shared instrument grants from the Bankhead-Coley Cancer Research program of the Florida Department of Health (06BS-02-9614 and 09BN-14). Project funding was received as subcontract from the Moffitt National Functional Genomics Center funded by the US Army Medical Research and Materiel Command under award DAMD17-02-2-0051. Amino acid analysis was performed by the Protein Chemistry Laboratory at Texas A&M University by Virginia Johnson, MS, and Larry Dangott, PhD.

Author information

Authors and Affiliations

  1. H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, FL, USA

    Yi Chen, Kate J. Fisher, Mark Lloyd, Elizabeth R. Wood, Domenico Coppola, Erin Siegel, David Shibata & Yian A. Chen

  2. Department of Surgery, University of Tennessee Health Science Center, Memphis, TN, USA

    David Shibata

  3. Molecular Oncology and Proteomics, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, 12902 Magnolia Drive, Tampa, FL, 33612, USA

    John M. Koomen

Authors
  1. Yi Chen
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  2. Kate J. Fisher
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  3. Mark Lloyd
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  4. Elizabeth R. Wood
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  5. Domenico Coppola
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  6. Erin Siegel
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  7. David Shibata
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  8. Yian A. Chen
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  9. John M. Koomen
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Corresponding author

Correspondence to John M. Koomen .

Editor information

Editors and Affiliations

  1. Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia, USA

    Iulia M. Lazar

  2. Department of Pharmaceutical Sciences, School of Pharmacy, Southern Illinois University Edwardsville, Edwardsville, Illinois, USA

    Maria Kontoyianni

  3. ImmunoGen, Waltham, Massachusetts, USA

    Alexandru C. Lazar

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Chen, Y. et al. (2017). Multiplexed Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry Quantification of Cancer Signaling Proteins. In: Lazar, I., Kontoyianni, M., Lazar, A. (eds) Proteomics for Drug Discovery. Methods in Molecular Biology, vol 1647. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7201-2_2

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  • DOI: https://doi.org/10.1007/978-1-4939-7201-2_2

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7200-5

  • Online ISBN: 978-1-4939-7201-2

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