Mass Spectrometry-Based Assay for Targeting Fifty-Two Proteins of Brain Origin in Cerebrospinal Fluid

Cerebrospinal fluid (CSF) is a circulatory fluid of the central nervous system and it can reflect the biochemical changes occurring in the brain. Although CSF retrieval through lumbar puncture is invasive, it remains the most commonly used fluid in exploring brain pathology as it is less complex and contains a higher concentration of brain-derived proteins than plasma (Reiber, H. Clin. Chim. Acta 2001, 310, 173−186; Macron et al. J. Proteome Res. 2018, 17, 4315−4319). We hypothesize that proteins produced by the brain will have diagnostic significance for brain pathologies. Hence, we expanded the previously in-house-developed 31-protein panel with more proteins classified as brain-specific by the Human Protein Atlas (HPA). Using the HPA, we selected 76 protein coding genes and screened CSF using liquid chromatography–mass spectrometry (LC–MS) and narrowed the protein list to candidates identified endogenously in CSF. Next, we developed a parallel reaction monitoring (PRM) assay for the 21 new proteins and merged it with the 31-protein assay developed earlier. In the process, we evaluated different screening strategies and optimized MS collision energies and ion isolation windows to achieve the highest possible analyte signal resulting in the PRM assay with an average linear dynamic range of 4.3 × 103. We also assessed the extent of Asn (N)–Gln (Q) deamidation, N-terminal pyro-Glu (E) conversion, and Met (M) oxidation and found that deamidation can be misassigned without high mass accuracy and high-resolution settings. We also assessed how many of these proteins could be reliably measured in 10 individual patient CSF samples. Our approach allows us to measure the relative levels of 52 brain-derived proteins in CSF by a single LC–MS method. This new assay may have important applications in discovering CSF biomarkers for various neurological diseases.脑脊液（Cerebrospinal fluid, CSF）是中枢神经系统的循环体液，可反映脑部发生的生化变化。尽管经腰椎穿刺获取脑脊液具有侵入性，但它仍是探索脑部病理状态最常用的体液样本，因其组分较血浆更为简单，且含有浓度更高的脑源性蛋白（Reiber, H. Clin. Chim. Acta 2001, 310, 173−186; Macron et al. J. Proteome Res. 2018, 17, 4315−4319）。我们提出假说：脑部产生的蛋白对脑部病理具有诊断价值。因此，我们在实验室自主开发的31蛋白检测面板基础上，新增了一批经人类蛋白质图谱（Human Protein Atlas, HPA）分类为脑特异性的蛋白。依托人类蛋白质图谱，我们筛选得到76个蛋白质编码基因，通过液相色谱-质谱联用法（liquid chromatography–mass spectrometry, LC–MS）对脑脊液样本进行检测，并将蛋白列表缩小至内源性存在于脑脊液中的候选蛋白。随后，我们针对21种新蛋白开发了平行反应监测（parallel reaction monitoring, PRM）检测方法，并将其与此前开发的31蛋白检测方法进行整合。在此过程中，我们评估了多种筛选策略，并优化了质谱碰撞能量与离子隔离窗口，以获得最优的分析物信号，最终建立的PRM检测方法的平均线性动态范围可达4.3×10³。我们还评估了天冬酰胺（Asn, N）-谷氨酰胺（Gln, Q）脱酰胺作用、N端焦谷氨酸（pyro-Glu, E）转化以及甲硫氨酸（Met, M）氧化的程度，发现若不采用高质量精度与高分辨率设置，脱酰胺作用可能被错误定性。我们同时评估了在10份患者个体脑脊液样本中可被可靠定量的蛋白数量。本研究方法可通过单次LC-MS检测，实现脑脊液中52种脑源性蛋白的相对定量。这一新型检测方法在发掘各类神经系统疾病的脑脊液生物标志物领域具有重要应用价值。