Table 1_Absolute quantification of tumor necrosis factor-alpha by isotope dilution mass spectrometry.docx

Tumor necrosis factor-α (TNF-α) 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-α based on liquid chromatography–isotope dilution tandem mass spectrometry (LC-IDMS) with amino acid or peptide analysis. The assays exhibited good linearity (R2 > 0.999), repeatability (RSD < 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-α could be fully hydrolyzed into amino acids after 60 h at 110 °C. The result based on the amino acid analysis was (0.770 ± 0.033) mg/g, expressed as a mass fraction (mg TNF-α per g of total solution), with an expanded uncertainty (k = 2). For peptide analysis, ANALLANGVELR (AR-12) and VVNLLSAIK (VK-9) were chosen as specific peptides. After 36 h of tryptic proteolysis, TNF-α could be completely proteolyzed into AR-12 and VK-9. Based on characteristic peptide analysis, the result was 0.769 ± 0.046 mg/g (k = 2). There was no significant difference between these two analyses, and the concentration of TNF-α was 0.770 ± 0.057 mg/g (k = 2), which was traceable to the International System of Units. Both methods developed in this study can accurately determine the concentration of TNF-α 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-α-induced cell apoptosis was significantly attenuated by antagonists, while detection kits based on three different principles exhibited good repeatability (RSD < 9%) and linearity (R2 > 0.999). This accurate, SI-traceable method can improve clinical TNF-α assay standardization and biomarker reliability.

肿瘤坏死因子-α（Tumor necrosis factor-α, TNF-α）是一类重要的炎症介质，在临床实践中已被广泛认可为多种自身免疫性疾病与感染性疾病的诊断生物标志物，例如类风湿关节炎。本研究基于液相色谱-同位素稀释串联质谱法（liquid chromatography–isotope dilution tandem mass spectrometry, LC-IDMS）结合氨基酸或肽段分析，建立了可溯源至国际单位制的TNF-α定量参考方法。该方法具有良好的线性（决定系数（Coefficient of Determination, R²）>0.999）、重复性（相对标准偏差（Relative Standard Deviation, RSD）<3%）与准确度，且通过有证参考物质（certified reference materials, CRMs）进行了验证。本研究选用3种稳定同位素标记氨基酸（缬氨酸、苯丙氨酸与亮氨酸）及2种肽段作为内标，以最小化测定变异。针对氨基酸分析路径，TNF-α可在110℃条件下水解60小时后完全转化为游离氨基酸；基于该路径得到的结果以质量分数（每克总溶液中TNF-α的毫克数）计为(0.770±0.033) mg/g，扩展不确定度为k=2。针对肽段分析路径，本研究选用ANALLANGVELR（AR-12）与VVNLLSAIK（VK-9）作为特征肽段。经36小时胰蛋白酶酶解后，TNF-α可完全被酶解为上述两种特征肽段；基于特征肽段分析得到的结果为(0.769±0.046) mg/g（k=2）。两种分析路径的结果无显著差异，最终TNF-α浓度为(0.770±0.057) mg/g（k=2），该结果可溯源至国际单位制。本研究开发的两种方法均可精准测定TNF-α浓度，可用于检测试剂盒开发与仪器校准。此外，本研究还提供了基于该方法认证的试剂在细胞凋亡实验与试剂盒评价中的应用数据：TNF-α诱导的细胞凋亡可被拮抗剂显著抑制，而基于三种不同原理的检测试剂盒均表现出良好的重复性（RSD<9%）与线性（R²>0.999）。本研究建立的精准且可溯源至国际单位制的方法，可推动临床TNF-α检测的标准化进程，并提升相关生物标志物检测的可靠性。