神经元特异性烯醇酶（NSE）是一种由αγ和γγ同工酶二聚体组成的有前途的小细胞肺癌（SCLC）生物标志物。由于传统的免疫测定存在干扰并且不能区分同工酶，我们开发了一种多重免疫亲和（IA）液相层析-串联质谱（LC-MS/MS）检测NSEα和NSEγ的方法，用于人类血清的定量分析。定标剂通过对重组表达的αα和γγ烯醇酶二聚体进行冷变性制备而成，诱导新的二聚体平衡态，大约为1αγ:1γγ:1αα。通过使用针对NSEγ特异性抗体进行IA萃取，实现了选择性样品纯化。所分离的αγ和γγ二聚体进行变性和胰蛋白酶消化，以便定量选定的标志性肽和其对应的同位素标记的内标准。得到的线性动态范围分别为1.5-56 ng/mL和0.64-167 ng/mL，对应于NSEα和NSEγ（R2分别为0.88和0.97）。检验结果表明，对NSEα和NSEγ的准确性和精密度均达到了可接受的水平。该方法成功地应用于患者血清中，检测到了两种同工酶。与传统的免疫测定相比，IA LC-MS/MS测得的总NSE浓度明显较低。通过这种多重IA LC-MS/MS检测方法，可以探索定量分析单个同工酶的临床价值。此外，与此处描述的定标剂一起，它可以应用于标准化不同平台的NSE免疫测定。版权所有 ©2023作者。由Elsevier B.V.发表。保留所有权利。

Neuron-specific enolase (NSE) is a promising small-cell lung cancer (SCLC) biomarker composed of αγ and γγ isozyme dimers. As the conventional immunoassays are prone to interferences and cannot differentiate between the isozymes, we developed a multiplex immunoaffinity (IA) liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the quantification of NSEα and NSEγ in human serum. A calibrator was prepared by performing cold denaturation of recombinantly expressed αα and γγ enolase dimers to induce a new dimer equilibrium that was determined to be approximately 1αγ:1γγ:1αα. Selective sample purification was achieved by performing IA extraction using an antibody specific towards NSEγ. The isolated αγ and γγ dimers were denatured and trypsin digested to allow quantification of the selected signature peptides and their corresponding isotopically labelled peptide internal standard. The obtained linear dynamic ranges were determined to be 1.5-56 ng/mL and 0.64-167 ng/mL for NSEα and NSEγ (R2 = 0.88 and 0.97 respectively). Validation of the assay showed acceptable accuracy and precision for NSEα and NSEγ. The method was successfully applied to patient serum in which both isozymes were detected. Compared to the conventional immunoassay, substantially lower total NSE concentrations were measured in IA LC-MS/MS. With this multiplex IA LC-MS/MS assay, the clinical value of quantifying the individual isozymes can be explored. In addition, together with the calibrator described here, it may be applied to standardize NSE immunoassays across different platforms.Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.