Abstract
Targeted quantitative mass spectrometry of immunoaffinity-enriched peptides, termed immuno-multiple reaction monitoring (iMRM), is a powerful method for determining the relative abundance of proteins in complex mixtures, like plasma or whole tissue. This technique combines 1,000-fold enrichment potential of antibodies for target peptides with the selectivity of multiple reaction monitoring mass spectrometry (MRM-MS). Using heavy isotope-labeled peptide counterparts as internal standards ensures high levels of precision. Further, LC-MRM-MS selectivity allows for multiplexing; antibodies recognizing different peptides can be added directly to a single mixture without subjecting to interferences common to other multiple antibody protein assays. Integrated extracted ion chromatograms (XIC) of product ions from endogenous unlabeled “light” peptide and stable isotope-labeled internal standard “heavy” peptides are used to generate a light/heavy peak area ratio. This ratio is proportional to the amount of peptide in the digestion mixture and can be used to estimate the concentration of protein in the sample.
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References
Addona T, Abbatiello SE, Schilling B, Skates SJ, Mani DR, Bunk DM, Spiegelman CH, Zimmerman LJ, Ham A-JL, Keshishian H, Hall SC, Allen S, Blackman RK, Borchers C, Buck CB, Cardasis HL, Cusack MP, Dodder NG, Gibson BW, Held JM, Hiltke T, Jackson A, Johansen EB, Kinsinger CR, Li J, Mesri M, Neubert TA, Niles RK, Pulsipher TC, Ransohoff D, Rodriguez H, Rudnick PA, Smith D, Tabb DL, Tegeler TJ, Variyath AM, Vega-Montoto LJ, Walhander A, Waldemarson S, Wang M, Whiteaker JR, Zhao L, Anderson NL, Fisher SJ, Liebler DC, Paulovich AG, Regnier FE, Tempst P, Carr SA (2009) Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma. Nat Biotechnol 27:633–641
Kuhn E, Addona T, Keshishian H, Burgess M, Mani DR, Lee RT, Sabatine MS, Gerszten RE, Carr SA (2009) Developing multiplexed assays for troponin I and interleukin 33 in plasma by peptide immunoaffinity enrichment and targeted mass spectrometry. Clin Chem 55:1108–1117
Kuhn E, Whiteaker JR, Mani DR, Jackson A, Zhao L, Pope ME, Smith D, Rivera KD, Anderson L, Skates SJ, Pearson TW, Paulovich AG, Carr SA (2012) Interlaboratory evaluation of automated multiplexed peptide immunoaffinity enrichment coupled to multiple reaction monitoring mass spectrometry for quantifying proteins in plasma. Mol Cell Proteomics 11:1–14
Ossola R, Schiess R, Picotti P, Rinner O, Reiter L, Aebersold R (2011) Biomarker validation in blood specimens by selected reaction monitoring mass spectrometry of N-glycosites. Methods Mol Biol 728:179–194
Picotti P, Aebersold R (2012) Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions. Nat Methods 9:555–566
Liebler DC, Zimmermann LJ (2013) Targeted quantitation of proteins by mass spectrometry. Biochemistry 52:3797–3806
Ebhardt HA (2014) Selected reaction monitoring mass spectrometry: a methodology overview. Methods Mol Biol 1072:209–222
Keshishian H, Addona T, Burgess M, Kuhn E, Carr SA (2007) Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution. Mol Cell Proteomics 12:2212–2229
Keshishian H, Addona T, Burgess M, Mani DR, Shi X, Kuhn E, Sabatine MS, Gerszten RE, Carr SA (2009) Quantification of cardiovascular biomarkers in patient plasma by targeted mass spectrometry and stable isotope dilution. Mol Cell Proteomics 10:2339–2349
Mani DR, Abbatiello SE, Carr SA (2012) Statistical characterization of multiple-reaction monitoring mass spectrometry (MRM-MS) assays for quantitative proteomics. BMC Bioinformatics 13:S9
Harlow E, Lane D (1999) Using antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 321–325
Anderson NL, Anderson NG, Haines LR, Hardie DB, Olafson RW, Pearson TW (2004) Mass spectrometric quantitation of peptides and proteins using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA). J Proteome Res 3:235–244
Whiteaker JR, Zhao L, Abbatiello SE, Burgess M, Kuhn E, Lim CW, Pope ME, Razavi M, Anderson NL, Pearson TW, Carr SA, Paulovich AJ (2011) Evaluation of large scale quantitative proteomics assay development using peptide affinity-based mass spectrometry. Mol Cell Proteomics 10:1–10
Schoenherr RM, Zhao L, Whiteaker JR, Feng L-C, Li L, Liu L, Liu X, Paulovich AG (2010) Automated screening of monoclonal antibodies for SISCAPA assays using a magnetic bead processor and liquid chromatography-selected reaction monitoring-mass spectrometry. J Immunol Methods 353(1–2):49–61
Deutsch EW, Lam H, Aebersold R (2008) PeptideAtlas: a resource for target selection for emerging targeted proteomics workflows. EMBO Rep 9:429–434
MacLean B, Tomazela DM, Shulman N, Chambers M, Gl F, Frewen B, Kern R, Tabb DL, Liebler DC, MacCoss MJ (2010) Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26:966–968
Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567
Fusaro VA, Mani DR, Mesirov JP, Carr SA (2009) Computational prediction of high responding peptides for development of targeted protein assays by mass spectrometry. Nat Biotechnol 27(2):190–198
Whiteaker JR, Zhao L, Lin C, Yan P, Wang P, Paulovich AG (2012) Sequential multiplexed analyte quantification using peptide immunoaffinity enrichment coupled to mass spectrometry. Mol Cell Proteomics. doi:10.1074/mcp.M111.015347
Acknowledgements
This work was partially financed through the Center for Translational Molecular Medicine, Breast CARE project (030-104-06).
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De Marchi, T., Kuhn, E., Carr, S.A., Umar, A. (2015). Antibody-Based Capture of Target Peptides in Multiple Reaction Monitoring Experiments. In: Vivanco, M. (eds) Mammary Stem Cells. Methods in Molecular Biology, vol 1293. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2519-3_7
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DOI: https://doi.org/10.1007/978-1-4939-2519-3_7
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