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Quantitative Mass Spectrometry by Isotope Dilution and Multiple Reaction Monitoring (MRM)

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Molecular Profiling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1606))

Abstract

Selected reaction monitoring (SRM) is used in molecular profiling to detect and quantify specific known proteins in complex mixtures. Using isotope dilution (Barnidge et al., Anal Chem 75(3):445–451, 2003) methodologies, peptides can be quantified without the need for an antibody-based method. Selected reaction monitoring assays employ electrospray ionization mass spectrometry (ESI-MS) followed by two stages of mass selection: a first stage where the mass of the peptide ion is selected and, after fragmentation by collision-induced dissociation (CID), a second stage (tandem MS) where either a single (e.g., SRM) or multiple (multiple reaction monitoring, MRM) specific peptide fragment ions are transmitted for detection. The MRM experiment is accomplished by specifying the parent masses of the selected endogenous and isotope-labeled peptides for MS/MS fragmentation and then monitoring fragment ions of interest, using their intensities/abundances and relative ratios to quantify the parent protein of interest. In this example protocol, we will utilize isotope dilution MRM-MS to quantify in absolute terms the total levels of the protein of interest, ataxia telangiectasia mutated (ATM) serine/threonine protein kinase. Ataxia telangiectasia mutated (ATM) phosphorylates several key proteins that initiate activation of the DNA damage checkpoint leading to cell cycle arrest.

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Acknowledgments

The authors are grateful to Dr. Lance Liotta, George Mason University, for expert editorial assistance.

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Authors and Affiliations

  1. Center for Applied Proteomics and Molecular Medicine, George Mason University, 10920 George Mason Circle MS1A9, Manassas, VA, 20110, USA

    Paul Russo

  2. DOD Gynecologic Cancer Center of Excellence, Annandale, VA, 22003, USA

    Brian L. Hood & Nicholas W. Bateman

  3. Inova Dwight and Martha Schar Cancer Institute, 3300 Gallows Road, Falls Church, VA, 22042, USA

    Thomas P. Conrads Ph.D.

  4. Gynecologic Cancer Center of Excellence, Women’s Health Integrated Research Center, 3289 Woodburn Rd, Suite 375, Annandale, VA, 22003, USA

    Thomas P. Conrads Ph.D.

Authors
  1. Paul Russo
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  2. Brian L. Hood
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  3. Nicholas W. Bateman
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  4. Thomas P. Conrads Ph.D.
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Corresponding authors

Correspondence to Paul Russo or Thomas P. Conrads Ph.D. .

Editor information

Editors and Affiliations

  1. Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, Virginia, USA

    Virginia Espina

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Russo, P., Hood, B.L., Bateman, N.W., Conrads, T.P. (2017). Quantitative Mass Spectrometry by Isotope Dilution and Multiple Reaction Monitoring (MRM). In: Espina, V. (eds) Molecular Profiling. Methods in Molecular Biology, vol 1606. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6990-6_20

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  • DOI: https://doi.org/10.1007/978-1-4939-6990-6_20

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6989-0

  • Online ISBN: 978-1-4939-6990-6

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