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Antibody-Based Capture of Target Peptides in Multiple Reaction Monitoring Experiments

  • Tommaso De Marchi
  • Eric Kuhn
  • Steven A. CarrEmail author
  • Arzu UmarEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1293)

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.

Key words

Targeted mass spectrometry MRM-MS Protein quantification Proteomics Immunoaffinity enrichment 

Notes

Acknowledgements

This work was partially financed through the Center for Translational Molecular Medicine, Breast CARE project (030-104-06).

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Copyright information

© Springer Science+Business Media LLC New York 2015

Authors and Affiliations

  1. 1.Department of Medical Oncology, Erasmus MC Cancer InstituteErasmus University Medical CenterRotterdamThe Netherlands
  2. 2.Postgraduate School of Molecular MedicineErasmus University Medical CenterRotterdamThe Netherlands
  3. 3.Broad Institute of MIT and HarvardCambridgeUSA
  4. 4.Center for Translational Molecular MedicineEindhovenThe Netherlands
  5. 5.Laboratory of Breast Cancer Genomics and Proteomics, Department of Medical Oncology, Erasmus MC Cancer InstituteErasmus University Medical CenterRotterdamThe Netherlands

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