Laboratory of Chemical Biology, Department of Biomedical Engineering and Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, the Netherlands; Catharina Hospital Eindhoven, Eindhoven, the Netherlands; Expert Center Clinical Chemistry Eindhoven, Eindhoven, the Netherlands.
Laboratory of Chemical Biology, Department of Biomedical Engineering and Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, the Netherlands.
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 May 15;1223:123701. doi: 10.1016/j.jchromb.2023.123701. Epub 2023 Apr 13.
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 (R = 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.
神经元特异性烯醇化酶（NSE）是一种有前途的小细胞肺癌（SCLC）标志物，由 αγ 和 γγ 同工酶二聚体组成。由于常规免疫测定容易受到干扰，并且无法区分同工酶，因此我们开发了一种用于定量人血清中 NSEα 和 NSEγ 的多重免疫亲和（IA）液相色谱-串联质谱（LC-MS/MS）测定法。通过对重组表达的αα和γγ烯醇酶二聚体进行冷变性来制备校准品，以诱导新的二聚体平衡，该平衡被确定为大约 1αγ:1γγ:1αα。通过使用针对 NSEγ 的抗体进行 IA 提取来实现选择性样品纯化。分离出的 αγ 和 γγ 二聚体变性并进行胰蛋白酶消化，以允许定量选择的特征肽及其相应的同位素标记肽内标。确定 NSEα 和 NSEγ 的获得线性动态范围分别为 1.5-56 ng/mL 和 0.64-167 ng/mL（R 分别为 0.88 和 0.97）。该测定的验证表明，NSEα 和 NSEγ 的准确性和精密度均可以接受。该方法已成功应用于可检测到两种同工酶的患者血清中。与常规免疫测定相比，IA LC-MS/MS 测量的总 NSE 浓度明显较低。使用这种多重 IA LC-MS/MS 测定法，可以探索定量单个同工酶的临床价值。此外，与本文所述的校准品一起，它可以应用于不同平台的 NSE 免疫测定标准化。
