A High-Accuracy Quantitative Method For Neurofilament Light Chain Protein

Alzheimer’s disease（AD）is a disease caused by the death or degeneration of nerve cells,which can lead to the gradual deterioration of memory,cognitive ability,motor function and other aspects,it brings huge burden and pressure to patients and families.The Neurofilament light chain（Nf L）is a highly expressed protein in neuronal axons,and its main function is to maintain and support the structure and stability of axons.Some studies have shown that Nf L protein is released to the surrounding environment when axons are damaged or die,so White Nf L levels in the blood and cerebrospinal fluid can be used to reflect the extent of neuronal damage and death in the brain,for the prediction and identification of AD.In recent years,the measurement technology for Nf L protein has rapidly advanced and improved.A series of highly sensitive immunoassay techniques,including Simoa technology,have been developed,which can detect lower levels of Nf L protein.Despite continuous advancements in measurement technology,accurately quantifying Nf L protein with high precision remains a challenge.The lack of a standardized quantification reference for Nf L protein has led to non-comparable results across different countries and laboratories.Therefore,this study establishes a high-precision absolute quantification method for Nf L based on Isotope Dilution Mass Spectrometry（IDMS）.（1）Establishing an absolute quantification method based on amino acids isotope dilution mass spectrometry（AA-based IDMS）.Proline,phenylalanine,leucine,and valine produced by Nf L protein acid hydrolysis,and their corresponding isotopically labeled amino acids were measured using liquid chromatography-mass spectrometry（LC-MS）with multiple reaction monitoring（MRM）to obtain accurate quantification.The hydrolysis time and hydrochloric acid addition,as well as the instrument parameters of LC-MS,were optimized during the measurement process.The final result selected for Nf L quantification was 218±4.73μg/g for Leu.（2）Establishing an absolute quantification method based on signature peptide isotope dilution mass spectrometry（Signature peptide-based ID-LCMS）.Three peptide segments FR-7（FASFIER）,AR-8（ALYEQEIR）,and FK-10（FTVLTESAAK）were screened and verified as characteristic peptide segments of Nf L,and then quantified using AA-based IDMS.Nf L was enzymatically cleaved and measured using LC-MS with MRM for three peptide segments corresponding to their isotopically labeled peptide segments.The enzymatic factors,peptide fragment sub-ions,and LC-MS instrument parameters were optimized.The average quantification results of the three peptide segments for Nf L were 217.49±5.56μg/g.（3）Establishing an absolute quantification method based on sulfur isotope dilution mass spectrometry（Sulfur-based ID-ICP-MS）.This method used high-performance liquid chromatography（HPLC）and high-resolution inductively coupled plasma mass spectrometry（HR-ICP-MS）in tandem,and used size-exclusion chromatography（SEC）to enrich and separate Nf L samples.³⁴S isotope dilution reagents were added after the column,and the ³²S and ³⁴S signal ratios were monitored.The Nf L quantification results were calculated based on the sulfur content.The final quantification result was215.58±1.00μg/g.By using three independent methods to accurately quantify the content of Nf L in simple samples,the total average result was 217.06±4.08μg/g.The results of the three methods were consistent,proving the accuracy and reliability of the IDMS-based method.This study laid a foundation for the development of Nf L standard substances and provided important references and guidance for the accurate quantification of NDD biomarkers.