Tau Protein Quantification in Human Cerebrospinal Fluid by Targeted Mass Spectrometry at High Sequence Coverage Provides Insights into Its Primary Structure Heterogeneity

Tau protein plays a major role in neurodegenerative disorders, appears to be a central biomarker of neuronal injury in cerebrospinal fluid (CSF), and is a promising target for Alzheimer’s disease immunotherapies. To quantify tau at high sensitivity and gain insights into its naturally occurring structural variations in human CSF, we coupled absolute quantification using protein standard with the multiplex detection capability of targeted high-resolution mass spectrometry (MS) on a Quadrupole-Orbitrap instrument. Using recombinant tau we developed a step-by-step workflow optimization including an extraction protocol that avoided affinity reagents and achieved the monitoring of 22 tau peptides uniformly distributed along the tau sequence. The lower limits of quantification ranged (LLOQ) from 150 to 1500 pg/mL depending on the peptide. Applied to endogenous CSF tau, up to 19 peptides were detected. Interestingly, there were significant differences in the abundance of peptides depending on their position in the sequence, with peptides from the tau mid-domain appearing significantly more abundant than peptides from the N- and C-terminus domains. This MS-based strategy provided results complementary to those of previous ELISA or Western Blot studies of CSF tau and could be applied to tau monitoring in human CSF cohorts.

tau蛋白（Tau protein）在神经退行性疾病中发挥关键作用，被认为是脑脊液（cerebrospinal fluid, CSF）中神经元损伤的核心生物标志物，同时也是阿尔茨海默病（Alzheimer’s disease）免疫治疗的潜在靶向靶点。为实现tau蛋白的高灵敏度定量，并解析其在人脑脊液中的天然结构变异，本研究将基于蛋白标准品的绝对定量技术，与四极杆-轨道阱质谱仪（Quadrupole-Orbitrap instrument）搭载的靶向高分辨率质谱（targeted high-resolution mass spectrometry, MS）多重检测能力相结合。研究人员以重组tau蛋白为材料，开发了一套分步优化的工作流程，其中包含无需亲和试剂的提取方案，可实现沿tau蛋白序列均匀分布的22个tau肽段的监测。根据肽段不同，定量下限（lower limits of quantification, LLOQ）范围为150至1500 pg/mL。将该方法应用于内源性脑脊液tau蛋白检测时，最多可检出19个肽段。值得注意的是，不同肽段的丰度与其在tau蛋白序列中的位置存在显著差异：tau蛋白中间结构域的肽段丰度显著高于N端与C端结构域的肽段。本研究建立的基于质谱的检测策略，与此前针对脑脊液tau蛋白的酶联免疫吸附试验（ELISA）、蛋白质印迹法（Western Blot）研究结果互为补充，可用于人脑脊液队列中的tau蛋白监测。