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