血浆Aβ42/Aβ40蛋白靶向在线富集与质谱检测策略及在阿尔兹海默病早期诊断中的研究

阿尔茨海默病（AD）是不可逆神经退行性疾病，需要早期诊断和干预。脑脊液Aβ42是AD重要早期诊断指标，但需要腰椎穿刺，患者接受程度低。最近研究表明血浆Aβ42/Aβ40比值对AD早期诊断更有价值。然而Aβ42和Aβ40结构相近且血浆中含量极低，临床精准检测存在技术困难。LC-MS/MS法是生物标志物检测的精准方法，有望实现血浆Aβ42/Aβ40比值精准检测，但需进一步提高灵敏度和自动化程度。本项目在前期工作基础上拟开展：①通过计算机辅助筛选技术设计构建特异性识别目标蛋白的多肽配基，开发高亲和聚合物整体柱；②构建新型二维液相串联质谱自动化“在线富集、分离、检测”平台实现血浆样本大体积直接进样，通过靶向在线富集和阀切换技术增加绝对进样量，获得灵敏度的大幅提高。③临床样本检测并系统分析该指标与经典指标相关性，为AD微创早筛提供重要的科学依据。本项目开展有望推动其他痕量蛋白精准富集和定量策略研究。

Alzheimer's disease (AD) is an irreversible neurodegenerative disease. Early diagnosis and intervention are the most effective strategies to delay the progression of AD. However, there is a lack of effective means of early diagnosis. Cerebro-Spinal Fluid (CSF) β-amyloid (Aβ) 42 is an important indicator for early diagnosis of AD, but lumbar puncture is required leading to low patient acceptance. This procedure is invasive and associated with the risks and uncertainties associated with lumbar puncture, resulting difficult to conduct large screening and studies. Recent studies have shown that the plasma Aβ42/Aβ40 ratio is more important in the early diagnosis of AD. However, Aβ42 and Aβ40 have similar structure and the very low concentration in plasma, so there are technical difficulties in simultaneous and accurate measurements, which limit the clinical application. The method based on isotopic diluent (ID)-Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) is recognized accurate for biomarker measurement, which is expected to achieve the accurate detection of plasma Aβ42/Aβ40 ratio, but it needs to further improve the sensitivity and automation. This project is planned to be carried out on the basis of the previous study: ①The peptide ligands for specific recognition of target proteins were designed and constructed by computer-aided screening technology, and the high affinity polymer monolithic column was developed to enrich target proteins in plasma. ②A new technology platform of "automatic online enrichment, separation and measurement" for two-dimensional LC-MS was established to achieve direct injection of plasma samples. The absolute injection volume was increased through targeted online enrichment and valve switching technology of large volume injection, and the sensitivity was greatly improved. ③Systematic analysis of the correlation between the index and the classical index, such as PET and CSF Aβ42 to provide an important scientific basis for AD non-invasive early screening. The development of this project is expected to promote the precise enrichment and quantitative strategy research of other low concentration proteins.