Coppens Salomé, Gogishvili Dea, Faustinelli Valentina, Scollo Emanuele, Hopley Christopher, Abeln Sanne, Dalby Paul, Goenaga-Infante Heidi, Luckau Luise, Vialaret Jérôme, Lehmann Sylvain, Hirtz Christophe, Illes-Toth Eva
National Measurement Laboratory, LGC, Queens Road, TW11 0LY Teddington, U.K.
PPC, IRMB-PPC, INM, Univ Montpellier, CHU Montpellier, INSERM CNRS, Montpellier 34295, France.
ACS Chem Neurosci. 2025 Jan 15;16(2):141-151. doi: 10.1021/acschemneuro.4c00526. Epub 2025 Jan 2.
Neurofilament light chain (NfL) is an early nonspecific biomarker in neurodegenerative diseases and traumatic brain injury, indicating axonal damage. This work describes the detailed structural characterization of a selected primary calibrator with the potential to be used in future reference measurement procedure (RMP) development for the accurate quantification of NfL. As a part of the described workflow, the sequence, higher-order structure as well as solvent accessibility, and hydrogen-bonding profile were assessed under three different conditions in KPBS, artificial cerebrospinal fluid, and artificial cerebrospinal fluid in the presence of human serum albumin. The results revealed that NfL is a structurally heterogeneous protein, eliciting a large conformational flexibility. Its structural ensemble changed when it was diluted with an aqueous buffer versus a surrogate matrix, artificial cerebrospinal fluid (aCSF), and/or aCSF with human serum albumin. Various regions of protection and deprotection in the protein head, central helical, and tail domains that experienced altered solvent accessibility and conformational changes caused by different solvent conditions were identified. Moreover, interfacial residues, which may play a role in a potential direct interaction between NfL and human serum albumin, emerged from hydrogen-deuterium exchange mass spectrometry (HDX-MS). These data pinpointed distinct regions of the protein that may participate in such an interaction. Overall, critical quality attributes of a potential primary calibrator for NfL measurements are provided. These findings will ultimately inform ongoing biochemical and clinical assay development procedures and manufacturing practices, giving careful consideration during sample handling and method development.
神经丝轻链（NfL）是神经退行性疾病和创伤性脑损伤中的一种早期非特异性生物标志物，表明轴突损伤。这项工作描述了一种选定的一级校准物的详细结构特征，该校准物有可能用于未来参考测量程序（RMP）的开发，以准确量化NfL。作为所述工作流程的一部分，在三种不同条件下，即在KPBS、人工脑脊液以及存在人血清白蛋白的人工脑脊液中，评估了序列、高阶结构以及溶剂可及性和氢键分布。结果表明，NfL是一种结构异质的蛋白质，具有很大的构象灵活性。当用含水缓冲液与替代基质、人工脑脊液（aCSF）和/或含人血清白蛋白的aCSF稀释时，其结构集合发生了变化。确定了蛋白质头部、中央螺旋和尾部结构域中由于不同溶剂条件而经历溶剂可及性改变和构象变化的各种保护和去保护区域。此外，氢氘交换质谱（HDX-MS）显示了可能在NfL与人血清白蛋白之间潜在直接相互作用中起作用的界面残基。这些数据确定了蛋白质中可能参与这种相互作用的不同区域。总体而言，提供了用于NfL测量的潜在一级校准物的关键质量属性。这些发现最终将为正在进行的生化和临床检测开发程序及生产实践提供信息，在样品处理和方法开发过程中需仔细考虑。
