{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T13:53:11Z","timestamp":1790949191416,"version":"4.1.0"},"reference-count":28,"publisher":"American Chemical Society (ACS)","issue":"10","license":[{"start":{"date-parts":[[2026,8,30]],"date-time":"2026-08-30T00:00:00Z","timestamp":1788048000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,8,30]],"date-time":"2026-08-30T00:00:00Z","timestamp":1788048000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,8,30]],"date-time":"2026-08-30T00:00:00Z","timestamp":1788048000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,10,2]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Antigen interference from residual antigen peptides is a persistent challenge in antipeptide immunocapture liquid chromatography\u2013tandem mass spectrometry (IC-LC\u2013MS\/MS) assays, particularly when quantifying low-abundance protein biomarkers. Traditionally, carrier protein-conjugated surrogate peptides identical to those used in LC\u2013MS\/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\u2013MS\/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\u2013MS\/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.<\/jats:p>","DOI":"10.1021\/acs.jproteome.6c00171","type":"journal-article","created":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T19:07:14Z","timestamp":1788376034000},"page":"5246-5254","source":"Crossref","is-referenced-by-count":0,"title":["A Novel Approach\nfor Generating Antigen Interference-Free\nAntipeptide Polyclonal Antibodies for Quantitative Measurements of\nLow-Abundance Protein Biomarkers Using Ultrasensitive Antipeptide\nImmunocapture LC\u2013MS\/MS Assays"],"prefix":"10.1021","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1643-7843","authenticated-orcid":true,"given":"Huidong","family":"Gu","sequence":"first","affiliation":[{"name":"Bioanalysis and Translational Mass Spectrometry, Bristol Myers Squibb , , ,","place":["Princeton, New Jersey, United States, 08543"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sagar","family":"Kawle","sequence":"additional","affiliation":[{"name":"Bioanalysis and Translational Mass Spectrometry, Bristol Myers Squibb , , ,","place":["Princeton, New Jersey, United States, 08543"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Suresh","family":"Venkateswaran","sequence":"additional","affiliation":[{"name":"Bioanalysis and Translational Mass Spectrometry, Bristol Myers Squibb , , ,","place":["Princeton, New Jersey, United States, 08543"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ana","family":"Liliac Zajontz","sequence":"additional","affiliation":[{"name":"Bioanalysis and Translational Mass Spectrometry, Bristol Myers Squibb , , ,","place":["Princeton, New Jersey, United States, 08543"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianing","family":"Zeng","sequence":"additional","affiliation":[{"name":"Bioanalytical and Material Sciences, Bristol Myers Squibb , , , ,","place":["Princeton, New Jersey, United States, 08543"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Graham","family":"Yearwood","sequence":"additional","affiliation":[{"name":"Bioanalytical and Material Sciences, Bristol Myers Squibb , , , ,","place":["Princeton, New Jersey, United States, 08543"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lina","family":"Luo","sequence":"additional","affiliation":[{"name":"Bioanalysis and Translational Mass Spectrometry, Bristol Myers Squibb , , ,","place":["Princeton, New Jersey, United States, 08543"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2000-598X","authenticated-orcid":true,"given":"Jim X.","family":"Shen","sequence":"additional","affiliation":[{"name":"Bioanalysis and Translational Mass Spectrometry, Bristol Myers Squibb , , ,","place":["Princeton, New Jersey, United States, 08543"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,30]]},"reference":[{"issue":"8","key":"2026100208412740600_cit1","doi-asserted-by":"publisher","first-page":"2411","DOI":"10.3390\/molecules27082411","article-title":"Applications of Tandem Mass Spectrometry (MS\/MS) in\nProtein Analysis for Biomedical Research","volume":"27","author":"Neagu","year":"2022","journal-title":"Molecules"},{"issue":"19","key":"2026100208412740600_cit2","doi-asserted-by":"publisher","first-page":"1203","DOI":"10.4155\/bio-2023-0129","article-title":"Quantitative bioanalysis of proteins\nby digestion and LC-MS\/MS: the use of multiple signature peptides","volume":"15","author":"Sleumer","year":"2023","journal-title":"Bioanalysis"},{"issue":"2","key":"2026100208412740600_cit3","doi-asserted-by":"publisher","first-page":"79","DOI":"10.1080\/17576180.2024.2415763","article-title":"Two decades\nof immunocapture liquid chromatography tandem mass spectrometry for\npharmaceutical discovery and development: reflections and recommendations\nfor optimal deployment","volume":"17","author":"Ackermann","year":"2025","journal-title":"Bioanalysis"},{"issue":"2","key":"2026100208412740600_cit4","doi-asserted-by":"publisher","first-page":"282","DOI":"10.1093\/clinchem\/hvz022","article-title":"Protein\nBiomarker Quantification by Immunoaffinity\nLiquid Chromatography\u2013Tandem Mass Spectrometry: Current State\nand Future Vision","volume":"66","author":"Neubert","year":"2020","journal-title":"Clin. Chem."},{"issue":"2","key":"2026100208412740600_cit5","doi-asserted-by":"publisher","first-page":"125","DOI":"10.1586\/14789450.2015.1024225","article-title":"Strategies\nfor improving sensitivity and selectivity\nfor the quantitation of biotherapeutics in biological matrix using\nLC-MS\/MS","volume":"12","author":"Shen","year":"2015","journal-title":"Expert Rev. Proteomics"},{"issue":"1","key":"2026100208412740600_cit6","doi-asserted-by":"publisher","first-page":"31","DOI":"10.1515\/revac-2013-0019","article-title":"Recent advances in\nabsolute quantification of peptides and proteins\nusing LC-MS","volume":"33","author":"Zhang","year":"2014","journal-title":"Rev. Anal. Chem."},{"issue":"3","key":"2026100208412740600_cit7","doi-asserted-by":"publisher","first-page":"FSO281","DOI":"10.4155\/fsoa-2017-0148","article-title":"Opportunities\nand challenges for hybrid immunoaffinity\nLC-MS approach for quantitative analysis of protein biomarkers","volume":"4","author":"Zhao","year":"2018","journal-title":"Future Sci. OA"},{"issue":"37","key":"2026100208412740600_cit8","doi-asserted-by":"publisher","first-page":"20303","DOI":"10.1021\/acs.analchem.5c03256","article-title":"An Antibody Cocktail-Based\nImmunoaffinity-LC-MS Method Enabled Ultra-Sensitive and Robust Quantification\nof Circulating Proinsulin Proteoforms and C-Peptide","volume":"97","author":"Shen","year":"2025","journal-title":"Anal. Chem."},{"issue":"21","key":"2026100208412740600_cit9","doi-asserted-by":"publisher","first-page":"14713","DOI":"10.1021\/acs.analchem.0c03271","article-title":"Antipeptide Immunocapture with In-Sample\nCalibration Curve Strategy for Sensitive and Robust LC-MS\/MS Bioanalysis\nof Clinical Protein Biomarkers in Formalin-Fixed Paraffin-Embedded\nTumor Tissues","volume":"92","author":"Zheng","year":"2020","journal-title":"Anal. Chem."},{"issue":"3","key":"2026100208412740600_cit10","doi-asserted-by":"publisher","first-page":"1719","DOI":"10.1021\/ac303031q","article-title":"Sequential Protein\nand Peptide Immunoaffinity Capture\nfor Mass Spectrometry-Based Quantification of Total Human Beta-Nerve\nGrowth Factor","volume":"85","author":"Neubert","year":"2013","journal-title":"Anal. Chem."},{"issue":"8","key":"2026100208412740600_cit11","doi-asserted-by":"publisher","first-page":"753","DOI":"10.4155\/bio-2015-0022","article-title":"Large-scale implementation of sequential\nprotein and peptide immunoaffinity enrichment LC\/nanoLC-MS\/MS for\nhuman \u03b2-nerve growth factor","volume":"8","author":"Schultz","year":"2016","journal-title":"Bioanalysis"},{"issue":"1","key":"2026100208412740600_cit12","doi-asserted-by":"publisher","first-page":"420","DOI":"10.1021\/acs.jproteome.7b00623","article-title":"Highly Reproducible\nAutomated Proteomics Sample Preparation Workflow for Quantitative\nMass Spectrometry","volume":"17","author":"Fu","year":"2018","journal-title":"J. Proteome Res."},{"issue":"16","key":"2026100208412740600_cit13","doi-asserted-by":"publisher","first-page":"8336","DOI":"10.1021\/ac501837t","article-title":"A Novel and Cost\nEffective Method of Removing Excess\nAlbumin from Plasma\/Serum Samples and Its Impacts on LC-MS\/MS Bioanalysis\nof Therapeutic Proteins","volume":"86","author":"Liu","year":"2014","journal-title":"Anal. Chem."},{"issue":"2","key":"2026100208412740600_cit14","doi-asserted-by":"publisher","first-page":"193","DOI":"10.3390\/ph17020193","article-title":"A Comprehensive\nImmunocapture-LC-MS\/MS Bioanalytical\nApproach in Support of a Biotherapeutic Ocular PK Study","volume":"17","author":"Chen","year":"2024","journal-title":"Pharmaceuticals (Basel)"},{"issue":"2","key":"2026100208412740600_cit15","doi-asserted-by":"publisher","first-page":"235","DOI":"10.1021\/pr034086h","article-title":"Mass Spectrometric Quantitation of Peptides and Proteins\nUsing Stable Isotope Standards and Capture by Anti-Peptide Antibodies\n(SISCAPA)","volume":"3","author":"Anderson","year":"2004","journal-title":"J. Proteome Res."},{"issue":"3","key":"2026100208412740600_cit16","doi-asserted-by":"publisher","first-page":"281","DOI":"10.4155\/bio.11.319","article-title":"Replacing immunoassays with tryptic digestion-peptide\nimmunoaffinity enrichment and LC-MS\/MS","volume":"4","author":"Becker","year":"2012","journal-title":"Bioanalysis"},{"key":"2026100208412740600_cit17","doi-asserted-by":"publisher","first-page":"25","DOI":"10.1007\/978-1-4939-6352-2_2","article-title":"Antibody Production\nwith Synthetic Peptides","volume":"1474","author":"Lee","year":"2016","journal-title":"Methods Mol. 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Neurosci."},{"issue":"6","key":"2026100208412740600_cit26","doi-asserted-by":"publisher","first-page":"1611","DOI":"10.1074\/mcp.M113.034140","article-title":"Immunoproteomics using polyclonal\nantibodies and stable isotope-labeled affinity-purified recombinant\nproteins","volume":"13","author":"Edfors","year":"2014","journal-title":"Mol. Cell. Proteomics"},{"issue":"7","key":"2026100208412740600_cit27","doi-asserted-by":"publisher","first-page":"1731","DOI":"10.1016\/j.cell.2018.10.014","article-title":"Anti-NKG2A mAb Is a\nCheckpoint Inhibitor that Promotes Anti-tumor Immunity by Unleashing\nBoth T and NK Cells","volume":"175","author":"Andre\u0301","year":"2018","journal-title":"Cell"},{"issue":"D1","key":"2026100208412740600_cit28","doi-asserted-by":"publisher","first-page":"D543","DOI":"10.1093\/nar\/gkae1011","article-title":"The PRIDE\ndatabase at 20 years: 2025 update","volume":"53","author":"Perez-Riverol","year":"2025","journal-title":"Nucleic Acids\nRes."}],"container-title":["Journal of Proteome Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.jproteome.6c00171","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jprobs\/article-pdf\/25\/10\/5246\/67868955\/acs.jproteome.6c00171.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jprobs\/article-pdf\/25\/10\/5246\/67868955\/acs.jproteome.6c00171.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T12:53:33Z","timestamp":1790945613000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jprobs\/article\/25\/10\/5246\/5371614\/A-Novel-Approach-for-Generating-Antigen"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,30]]},"references-count":28,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2026,8,30]]},"published-print":{"date-parts":[[2026,10,2]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.jproteome.6c00171","relation":{},"ISSN":["1535-3893","1535-3907"],"issn-type":[{"value":"1535-3893","type":"print"},{"value":"1535-3907","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,30]]}}}