{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T02:13:56Z","timestamp":1789438436190,"version":"build-2803163510"},"reference-count":35,"publisher":"American Chemical Society (ACS)","issue":"21","license":[{"start":{"date-parts":[[2020,10,13]],"date-time":"2020-10-13T00:00:00Z","timestamp":1602547200000},"content-version":"vor","delay-in-days":22,"URL":"https:\/\/pubs.acs.org\/page\/policy\/authorchoice_termsofuse.html"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2020,11,3]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Despite huge promises, bioanalysis of protein biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues by liquid chromatography-tandem mass spectrometry (LC-MS\/MS) for clinical applications is still very challenging. Here, we describe a sensitive and robust LC-MS\/MS assay to quantify clinical protein biomarkers in FFPE tumor sections using automated antipeptide antibody immunocapture followed by in-sample calibration curve (ISCC) strategy with multiple isotopologue reaction monitoring (MIRM) technique. ISCC approach with MIRM of stable isotopically labeled (SIL) peptides eliminated the need for authentic matrices for external calibration curves, overcame the matrix effects, and validated the quantification range in each individual sample. Specifically, after deparaffinization, rehydration, antigen retrieval, and homogenization, the protein analytes in FFPE tumor tissues were spiked with a known concentration of one SIL peptide for each analyte, followed by trypsin digestion and antipeptide immunocapture enrichment prior to MIRM-ISCC-based LC-MS\/MS analysis. This approach has been successfully used for sensitive quantification of programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) in 15 representative FFPE tumor samples from lung, colorectal, and head and neck cancer patients. Except for one sample, PD-L1 and PD-1 in all samples were quantifiable using this assay with concentrations of 27.85\u2013798.43 (amol\/\u03bcg protein) for PD-L1 and 16.96\u2013129.89 (amol\/\u03bcg protein) for PD-1. These results were generally in agreement with the immunohistochemistry (IHC) data but with some exceptions. This approach demonstrated the feasibility to quantify low abundant protein biomarkers in FFPE tissues with improved sensitivity, specificity, and robustness and showed great potential as an orthogonal analytical approach to IHC for clinical applications.<\/jats:p>","DOI":"10.1021\/acs.analchem.0c03271","type":"journal-article","created":{"date-parts":[[2020,9,21]],"date-time":"2020-09-21T11:49:16Z","timestamp":1600688956000},"page":"14713-14722","source":"Crossref","is-referenced-by-count":23,"title":["Antipeptide Immunocapture with In-Sample Calibration\nCurve Strategy for Sensitive and Robust LC-MS\/MS Bioanalysis of Clinical\nProtein Biomarkers in Formalin-Fixed Paraffin-Embedded Tumor Tissues"],"prefix":"10.1021","volume":"92","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5121-9024","authenticated-orcid":true,"given":"Naiyu","family":"Zheng","sequence":"first","affiliation":[{"name":"Bristol Myers Squibb , , Route 206 and Province Line Road , , ,","place":["Princeton, New Jersey, United States, 08543"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kristin","family":"Taylor","sequence":"additional","affiliation":[{"name":"Bristol Myers Squibb , , Route 206 and Province Line Road , , ,","place":["Princeton, New Jersey, United States, 08543"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1643-7843","authenticated-orcid":true,"given":"Huidong","family":"Gu","sequence":"additional","affiliation":[{"name":"Bristol Myers Squibb , , Route 206 and Province Line Road , , ,","place":["Princeton, New Jersey, United States, 08543"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5610-9051","authenticated-orcid":true,"given":"Rasa","family":"Santockyte","sequence":"additional","affiliation":[{"name":"Bristol Myers Squibb , , Route 206 and Province Line Road , , ,","place":["Princeton, New Jersey, United States, 08543"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xi-Tao","family":"Wang","sequence":"additional","affiliation":[{"name":"Bristol Myers Squibb , , Route 206 and Province Line Road , , ,","place":["Princeton, New Jersey, United States, 08543"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jean","family":"McCarty","sequence":"additional","affiliation":[{"name":"Bristol Myers Squibb , , Route 206 and Province Line Road , , ,","place":["Princeton, New Jersey, United States, 08543"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Olufemi","family":"Adelakun","sequence":"additional","affiliation":[{"name":"Bristol Myers Squibb , , Route 206 and Province Line Road , , ,","place":["Princeton, New Jersey, United States, 08543"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yan J.","family":"Zhang","sequence":"additional","affiliation":[{"name":"Bristol Myers Squibb , , Route 206 and Province Line Road , , ,","place":["Princeton, New Jersey, United States, 08543"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Renuka","family":"Pillutla","sequence":"additional","affiliation":[{"name":"Bristol Myers Squibb , , Route 206 and Province Line Road , , ,","place":["Princeton, New Jersey, United States, 08543"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianing","family":"Zeng","sequence":"additional","affiliation":[{"name":"Bristol Myers Squibb , , Route 206 and Province Line Road , , ,","place":["Princeton, New Jersey, United States, 08543"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2020,9,21]]},"reference":[{"key":"2026090117045644000_cit1","doi-asserted-by":"publisher","first-page":"341","DOI":"10.5858\/arpa.2015-0506-SA","article-title":"Members of Pulmonary Pathology, S., Programmed Death\nLigand-1 Immunohistochemistry--A New Challenge for Pathologists: A\nPerspective From Members of the Pulmonary Pathology Society","volume":"140","author":"Sholl","year":"2016","journal-title":"Arch. Pathol. Lab. Med."},{"key":"2026090117045644000_cit2","first-page":"289","article-title":"An Introduction to the Performance\nof Immunohistochemistry","volume-title":"Methods in Molecular\nBiology","author":"Magaki","year":"2019"},{"key":"2026090117045644000_cit3","first-page":"115","article-title":"Principles and Methods of Immunohistochemistry","volume-title":"Methods in Molecular Biology","author":"Ramos-Vara","year":"2017"},{"key":"2026090117045644000_cit4","doi-asserted-by":"publisher","first-page":"839","DOI":"10.1074\/mcp.RA119.001889","article-title":"Quantitative profiling\nof the human\nsubstantia nigra proteome from laser-capture microdissected FFPE tissue","volume":"19","author":"Griesser","year":"2020","journal-title":"Mol. Cell. Proteomics"},{"key":"2026090117045644000_cit5","doi-asserted-by":"publisher","DOI":"10.1002\/prca.201700167","article-title":"On-Tissue\nHydrogel-Mediated Nondestructive Proteomic Characterization: Application\nto fr\/fr and FFPE Tissues and Insights for Quantitative Proteomics\nUsing a Case of Cardiac Myxoma","volume":"13","author":"Taverna","year":"2019","journal-title":"Proteomics:\nClin. Appl."},{"key":"2026090117045644000_cit6","doi-asserted-by":"publisher","DOI":"10.1186\/1559-0275-11-28","article-title":"Critical comparison\nof sample preparation strategies\nfor shotgun proteomic analysis of formalin-fixed, paraffin-embedded\nsamples: insights from liver tissue","volume":"11","author":"Tanca","year":"2014","journal-title":"Clin. Proteomics"},{"key":"2026090117045644000_cit7","doi-asserted-by":"publisher","first-page":"1705","DOI":"10.1074\/mcp.RA117.000037","article-title":"Quantitative Mass Spectrometry Analysis\nof PD-L1 Protein Expression, N-glycosylation and Expression Stoichiometry\nwith PD-1 and PD-L2 in Human Melanoma","volume":"16","author":"Morales-Betanzos","year":"2017","journal-title":"Mol.\nCell. Proteomics"},{"key":"2026090117045644000_cit8","doi-asserted-by":"publisher","first-page":"3498","DOI":"10.1021\/pr300130t","article-title":"Precision of multiple\nreaction monitoring mass spectrometry analysis of formalin-fixed,\nparaffin-embedded tissue","volume":"11","author":"Sprung","year":"2012","journal-title":"J. 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Proteome Res."},{"key":"2026090117045644000_cit10","doi-asserted-by":"publisher","first-page":"2623","DOI":"10.1002\/pmic.201400110","article-title":"Integrated\nand convenient procedure for protein extraction\nfrom formalin-fixed, paraffin-embedded tissues for LC-MS\/MS analysis","volume":"14","author":"Lai","year":"2014","journal-title":"Proteomics"},{"key":"2026090117045644000_cit11","doi-asserted-by":"publisher","first-page":"2717","DOI":"10.1021\/acs.jproteome.6b00245","article-title":"Optimized\nProtocol for Quantitative\nMultiple Reaction Monitoring-Based Proteomic Analysis of Formalin-Fixed,\nParaffin-Embedded Tissues","volume":"15","author":"Kennedy","year":"2016","journal-title":"J. Proteome Res."},{"key":"2026090117045644000_cit12","doi-asserted-by":"publisher","first-page":"426","DOI":"10.1021\/acs.jproteome.8b00699","article-title":"Data-Independent\nAcquisition Mass Spectrometry To Quantify Protein Levels in FFPE Tumor\nBiopsies for Molecular Diagnostics","volume":"18","author":"Kim","year":"2019","journal-title":"J. Proteome\nRes."},{"key":"2026090117045644000_cit13","doi-asserted-by":"publisher","first-page":"3932","DOI":"10.1021\/acs.jproteome.8b00605","article-title":"Multiplex\nImmuno-Liquid Chromatography-Mass\nSpectrometry-Parallel Reaction Monitoring (LC-MS-PRM) Quantitation\nof CD8A, CD4, LAG3, PD1, PD-L1, and PD-L2 in Frozen Human Tissues","volume":"17","author":"Zhang","year":"2018","journal-title":"J. Proteome Res."},{"key":"2026090117045644000_cit14","doi-asserted-by":"publisher","first-page":"29","DOI":"10.1016\/j.canlet.2017.06.033","article-title":"PD-1\/PD-L1\ncheckpoint blockades in non-small cell lung cancer: New development\nand challenges","volume":"405","author":"Meng","year":"2017","journal-title":"Cancer Lett."},{"key":"2026090117045644000_cit15","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1177\/1758835918763493","article-title":"PD-L1 expression\ntesting\nin non-small cell lung cancer","volume":"10","author":"Teixido\u0301","year":"2018","journal-title":"Ther. Adv. Med.\nOncol."},{"key":"2026090117045644000_cit16","doi-asserted-by":"publisher","DOI":"10.3390\/molecules24061190","article-title":"Molecular\nInteractions of Antibody\nDrugs Targeting PD-1, PD-L1, and CTLA-4 in Immuno-Oncology","volume":"24","author":"Lee","year":"2019","journal-title":"Molecules"},{"key":"2026090117045644000_cit17","doi-asserted-by":"publisher","DOI":"10.1186\/s40425-016-0153-x","article-title":"PD-L1 biomarker\ntesting for non-small cell lung cancer:\ntruth or fiction?","volume":"4","author":"Grigg","year":"2016","journal-title":"J Immunother. Cancer"},{"key":"2026090117045644000_cit18","doi-asserted-by":"publisher","first-page":"32","DOI":"10.1016\/j.coph.2015.05.011","article-title":"PD-1\/PD-L1 inhibitors","volume":"23","author":"Sunshine","year":"2015","journal-title":"Curr.\nOpin. 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Chem."},{"key":"2026090117045644000_cit24","doi-asserted-by":"publisher","first-page":"385","DOI":"10.1016\/j.cll.2011.07.004","article-title":"Peptide\nimmunoaffinity enrichment coupled with mass\nspectrometry for peptide and protein quantification","volume":"31","author":"Whiteaker","year":"2011","journal-title":"Clin. Lab. Med."},{"key":"2026090117045644000_cit25","doi-asserted-by":"publisher","first-page":"1066","DOI":"10.1007\/s10120-015-0566-0","article-title":"Mass-spectrometry-based\nquantitation of Her2 in gastroesophageal tumor tissue: comparison\nto IHC and FISH","volume":"19","author":"Catenacci","year":"2016","journal-title":"Gastric Cancer"},{"key":"2026090117045644000_cit26","doi-asserted-by":"publisher","first-page":"4934","DOI":"10.1021\/acs.analchem.8b05802","article-title":", Isotopologue Multipoint\nCalibration\nfor Proteomics Biomarker Quantification in Clinical Practice","volume":"91","author":"Chiva","year":"2019","journal-title":"Anal. Chem."},{"key":"2026090117045644000_cit27","doi-asserted-by":"publisher","first-page":"2536","DOI":"10.1021\/acs.analchem.8b05656","article-title":"In-Sample\nCalibration Curve Using\nMultiple Isotopologue Reaction Monitoring of a Stable Isotopically\nLabeled Analyte for Instant LC-MS\/MS Bioanalysis and Quantitative\nProteomics","volume":"91","author":"Gu","year":"2019","journal-title":"Anal. Chem."},{"key":"2026090117045644000_cit28","doi-asserted-by":"publisher","first-page":"8652","DOI":"10.1021\/acs.analchem.9b02136","article-title":"Eliminating Preparation of Multisample\nExternal Calibration Curves and Dilution of Study Samples Using the\nMultiple Isotopologue Reaction Monitoring (MIRM) Technique in Quantitative\nLC-MS\/MS Bioanalysis","volume":"91","author":"Gu","year":"2019","journal-title":"Anal. Chem."},{"key":"2026090117045644000_cit29","doi-asserted-by":"publisher","first-page":"205","DOI":"10.1007\/s00726-013-1494-0","article-title":"Analysis of\nthe formalin-fixed paraffin-embedded\ntissue proteome: pitfalls, challenges, and future prospectives","volume":"45","author":"Maes","year":"2013","journal-title":"Amino Acids"},{"key":"2026090117045644000_cit30","doi-asserted-by":"publisher","first-page":"896","DOI":"10.1021\/pr100812d","article-title":"Initial\ndevelopment and validation of a novel extraction method for quantitative\nmining of the formalin-fixed, paraffin-embedded tissue proteome for\nbiomarker investigations","volume":"10","author":"Nirmalan","year":"2011","journal-title":"J. Proteome Res."},{"key":"2026090117045644000_cit31","doi-asserted-by":"publisher","first-page":"859","DOI":"10.4155\/bio.14.36","article-title":"Application\nand challenges in using LC-MS assays for absolute quantitative analysis\nof therapeutic proteins in drug discovery","volume":"6","author":"Zheng","year":"2014","journal-title":"Bioanalysis"},{"key":"2026090117045644000_cit32","doi-asserted-by":"publisher","first-page":"382","DOI":"10.1074\/mcp.O114.043133","article-title":"Anti-peptide monoclonal antibodies generated for immuno-multiple\nreaction monitoring-mass spectrometry assays have a high probability\nof supporting Western blot and ELISA","volume":"14","author":"Schoenherr","year":"2015","journal-title":"Mol. Cell.\nProteomics"},{"key":"2026090117045644000_cit33","doi-asserted-by":"publisher","first-page":"47","DOI":"10.1016\/j.dnarep.2018.03.003","article-title":"Targeted\nmass spectrometry enables\nrobust quantification of FANCD2 mono-ubiquitination in response to\nDNA damage","volume":"65","author":"Whiteaker","year":"2018","journal-title":"DNA Repair"},{"key":"2026090117045644000_cit34","doi-asserted-by":"publisher","first-page":"19","DOI":"10.1038\/nmeth.2285","article-title":"Targeted proteomics","volume":"10","author":"Marx","year":"2013","journal-title":"Nat. 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