A Novel Approach for Generating Antigen Interference-Free Antipeptide Polyclonal Antibodies for Quantitative Measurements of Low-Abundance Protein Biomarkers Using Ultrasensitive Antipeptide Immunocapture LC–MS/MS Assays

Abstract Antigen interference from residual antigen peptides is a persistent challenge in antipeptide immunocapture liquid chromatography–tandem mass spectrometry (IC-LC–MS/MS) assays, particularly when quantifying low-abundance protein biomarkers. Traditionally, carrier protein-conjugated surrogate peptides identical to those used in LC–MS/MS quantification are employed as antigens for immunization, often resulting in varying degrees of antigen interference from the resulting antipeptide polyclonal antibodies (pAbs). While precapture purification and other mitigation strategies can reduce interference, they may be insufficient for ultrasensitive antipeptide IC-LC–MS/MS assays. Here, for the first time, we demonstrate the use of carrier-protein-conjugated, stable isotopically labeled surrogate peptides as antigens for immunization. This strategy ensures that residual antigen peptide is distinguishable from endogenous analytes by mass spectrometry, effectively eliminating antigen interference. This paper details the design principles for stable isotopically labeled surrogate peptides used in antigen preparation and compares the performance of pAbs generated using labeled versus traditional nonlabeled peptide antigens. Results from over 20 batches of antipeptide pAbs demonstrate that this method enables the routine generation of antigen interference-free antipeptide pAbs, facilitating the development of ultrasensitive antipeptide IC-LC–MS/MS assays for low-abundance protein biomarker quantification without incurring additional time and significant cost. Data are available via ProteomeXchange with an identifier of PXD076272.