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，被视作脑脊液（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免疫治疗的极具前景的靶点。为实现tau蛋白的高灵敏度定量，并深入解析其在人类脑脊液中天然存在的结构变异，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（Quadrupole-Orbitrap）质谱仪所具备的靶向高分辨率质谱（mass spectrometry, MS）多重检测能力相结合。本研究以重组tau蛋白为材料，开发了一套分步优化的实验工作流程，其中包含无需亲和试剂的提取方案，实现了对tau蛋白序列上均匀分布的22条tau肽段的靶向监测。根据肽段的不同，其定量下限（lower limit 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蛋白的检测时，最多可检出19条肽段。值得注意的是，肽段的丰度会随其在tau蛋白序列中的位置呈现显著差异：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。这种基于质谱的检测策略所得结果与既往针对脑脊液tau蛋白的酶联免疫吸附测定（enzyme-linked immunosorbent assay, ELISA）、蛋白质免疫印迹（Western Blot）研究结果互补，可用于人类脑脊液队列的tau蛋白监测。