Multiplexed Immunoaffinity Enrichment of Peptides with Anti-peptide Antibodies and Quantification by Stable Isotope Dilution Multiple Reaction Monitoring Mass Spectrometry

  • Eric KuhnEmail author
  • Steven A. CarrEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1410)


Immunoaffinity enrichment of peptides using anti-peptide antibodies and their subsequent analysis by targeted mass spectrometry using stable isotope-labeled peptide standards is a sensitive and relatively high-throughput assay technology for unmodified and modified peptides in cells, tissues, and biofluids. Suppliers of antibodies and peptides are increasingly aware of this technique and have started incorporating customized quality measures and production protocols to increase the success rate, performance, and supply of the necessary reagents. Over the past decade, analytical biochemists, clinical diagnosticians, antibody experts, and mass spectrometry specialists have shared ideas, instrumentation, reagents, and protocols, to demonstrate that immuno-MRM-MS is reproducible across laboratories. Assay performance is now suitable for verification of candidate biomarkers from large scale discovery “omics” studies, measuring diagnostic proteins in plasma in the clinical laboratory, and for developing a companion assay for preclinical drug studies. Here we illustrate the process for developing these assays with a step-by-step guide for a 20-plex immuno-MRM-MS assay. We emphasize the need for analytical validation of the assay to insure that antibodies, peptides, and mass spectrometer are working as intended, in a multiplexed manner, with suitable assay performance (median values for 20 peptides: CV = 12.4 % at 740 amol/μL, LOD = 310 amol/μL) for applications in quantitative biology and candidate biomarker verification. The assays described conform to Tier 2 (of 3) level of analytical assay validation (1), meaning that the assays are capable of repeatedly measuring sets of analytes of interest within and across samples/experiments and employ internal standards for each analyte for confident detection and precise quantification.

Key words

Anti-peptide antibody Protein assay Peptide assay Multiplexed Quantification Mass spectrometry Immunoaffinity enrichment Reverse curves Plasma Biomarkers Multiple reaction monitoring Selected reaction monitoring Parallel reaction monitoring 



This work was supported in part by grants from National Institutes of Health: HHSN268201000033C and R01HL096738 from NHLBI and Grants U24CA160034 from NCI Clinical Proteomics Tumor Analysis Consortium initiative and 5U01CA152990-05 from the NCI Early Detection Research Network program (to SAC).


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Broad Institute of MIT and HarvardCambridgeUSA

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