The original data of the manuscript for the Absolute quantification of tumor necrosis factor-alpha by isotope dilution mass spectrometry

TNF-α is an important indicator for the auxiliary diagnosis of inflammation and malignant tumors, and its accurate quantification helps ensurethe accuracy and effectiveness of diagnosis and treatment for related diseases. However, immunological methods for detecting TNF-α rely onspecific antibodies and exhibit low sensitivity and batch-to-batch variability. Therefore, we established isotope dilution mass spectrometry?based methods for quantifying tumor necrosis factor TNF-α, utilizing both amino acid analysis and characteristic peptide analysis. Rawmaterial validation included molecular weight verification, purity analysis, affinity experiments, different TNF-α in vitro detection kits, and TNF-α-induced apoptosis experiments. The methodological levels of the two established methods were also examined to ensure the accuracy ofquantification. The methods are traceable to the International System of Units (SI), which facilitates absolute protein quantification, kitdevelopment, and instrument calibration. The results indicated that the TNF-α protein has high purity and physiological activity, making it asuitable subject for quantitative analysis. The amino acid-based TNF-α quantification results were (0.770 ± 0.033) mg g-1 (k = 2), and thecharacteristic peptide-based TNF-α quantification results were (0.769 ± 0.046) mg g-1 (k = 2). There was no significant difference betweenthe two quantification results.