{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T13:00:37Z","timestamp":1785330037270,"version":"3.55.0"},"reference-count":29,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,2,6]],"date-time":"2026-02-06T00:00:00Z","timestamp":1770336000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Chem."],"abstract":"<jats:p>\n                    Tumor necrosis factor-\u03b1 (TNF-\u03b1) is an important inflammatory mediator and has been widely recognized as a diagnostic biomarker for various autoimmune and infectious diseases in clinical practice, such as rheumatoid arthritis. In this study, we established an SI-traceable reference method to quantify TNF-\u03b1 based on liquid chromatography\u2013isotope dilution tandem mass spectrometry (LC-IDMS) with amino acid or peptide analysis. The assays exhibited good linearity (\n                    <jats:italic>R<\/jats:italic>\n                    <jats:sup>2<\/jats:sup>\n                    &amp;gt; 0.999), repeatability (RSD &amp;lt; 3%) and accuracy, which had been verified using certified reference materials (CRMs). Stable isotope-labeled version of three amino acids (valine, phenylalanine, and leucine) and two peptides were used as an internal standard to minimize assay variability. For amino acid analysis, TNF-\u03b1 could be fully hydrolyzed into amino acids after 60\u00a0h at 110\u00a0\u00b0C. The result based on the amino acid analysis was (0.770 \u00b1 0.033) mg\/g, expressed as a mass fraction (mg TNF-\u03b1 per g of total solution), with an expanded uncertainty (\n                    <jats:italic>k<\/jats:italic>\n                    = 2). For peptide analysis, ANALLANGVELR (AR-12) and VVNLLSAIK (VK-9) were chosen as specific peptides. After 36\u00a0h of tryptic proteolysis, TNF-\u03b1 could be completely proteolyzed into AR-12 and VK-9. Based on characteristic peptide analysis, the result was 0.769 \u00b1 0.046 mg\/g (\n                    <jats:italic>k = 2<\/jats:italic>\n                    ). There was no significant difference between these two analyses, and the concentration of TNF-\u03b1 was 0.770 \u00b1 0.057 mg\/g (\n                    <jats:italic>k = 2<\/jats:italic>\n                    ), which was traceable to the International System of Units. Both methods developed in this study can accurately determine the concentration of TNF-\u03b1 and are useful for detection kit development and instrument calibration. In addition, application data for reagents certified by these methods in cell apoptosis assays and kit evaluation are provided: TNF-\u03b1-induced cell apoptosis was significantly attenuated by antagonists, while detection kits based on three different principles exhibited good repeatability (RSD &amp;lt; 9%) and linearity (\n                    <jats:italic>R<\/jats:italic>\n                    <jats:sup>2<\/jats:sup>\n                    &amp;gt; 0.999). This accurate, SI-traceable method can improve clinical TNF-\u03b1 assay standardization and biomarker reliability.\n                  <\/jats:p>","DOI":"10.3389\/fchem.2025.1667885","type":"journal-article","created":{"date-parts":[[2026,2,6]],"date-time":"2026-02-06T10:46:01Z","timestamp":1770374761000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":1,"title":["Absolute quantification of tumor necrosis factor-alpha by isotope dilution mass spectrometry"],"prefix":"10.3389","volume":"13","author":[{"given":"Chenchen","family":"Bao","sequence":"first","affiliation":[{"name":"Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University","place":["Hangzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing","family":"Wang","sequence":"additional","affiliation":[{"name":"Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University","place":["Hangzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenqiang","family":"Xia","sequence":"additional","affiliation":[{"name":"Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University","place":["Hangzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ke","family":"Yang","sequence":"additional","affiliation":[{"name":"Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University","place":["Hangzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinchang","family":"Shi","sequence":"additional","affiliation":[{"name":"NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, National Institute for Food and Drug Control","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoying","family":"Yang","sequence":"additional","affiliation":[{"name":"Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University","place":["Hangzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hailin","family":"Li","sequence":"additional","affiliation":[{"name":"Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University","place":["Hangzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuemin","family":"Wang","sequence":"additional","affiliation":[{"name":"Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University","place":["Hangzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yafen","family":"Zhang","sequence":"additional","affiliation":[{"name":"Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University","place":["Hangzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,2,6]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"198","DOI":"10.1016\/j.ics.2004.02.087","article-title":"Mass spectrometry-based proteomics","volume":"422","author":"Aebersold","year":"2003","journal-title":"Nature"},{"key":"B22","doi-asserted-by":"publisher","first-page":"361","DOI":"10.1038\/nrc2628","article-title":"Tumour necrosis factor and cancer","volume":"9","author":"Balkwill","year":"2009","journal-title":"Nat. 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