A novel multi-faceted method for large-scale characterization and relative quantitation of citrullinated proteins for biological samples and its application to Alzheimer's disease

摘要 蛋白质瓜氨酸化作为一种酶促翻译后修饰(PTM)在糖尿病的发生和发展中起着重要的作用。 许多疾病的进展，包括类风湿性关节炎、神经退行性疾病、糖尿病和 心血管问题。目前对瓜氨酸的研究主要依赖于免疫组织化学和 Western blotting技术，缺乏有效的定性和定量分析方法 临床相关样本中的瓜氨酸蛋白，限制了我们对这种疾病相关PTM的了解。质量 光谱学(MS)已成为蛋白质组学和许多PTM的位点特异性分析的强大工具，包括 乙酰化、磷酸化和糖基化。然而，基于MS的瓜氨酸化分析显示 由于缺乏有效的方法在生物中特异地浓缩瓜氨酸蛋白而带来的挑战 样品，以及基于串联-MS的方法，使得能够准确地鉴定瓜氨酸多肽和可靠的 瓜氨酸化部位的指定。这项提议的主要目标是开发一种多方面的方法 将生物素标记辅助富集法、基于等压标记的定量策略与MS-MS相结合 基于同时浓缩、大规模表征和相对定量的技术 生物样品中的瓜氨酸蛋白，并应用这一多方面的方法来研究 瓜氨酸化在阿尔茨海默病中的作用。我们提出以下具体目标：具体目标1- 建立一种新的基于生物素标记物辅助MS的瓜氨酸蛋白大规模表征方法 在生物样本中。具体目标2-建立一种多方面的方法，集成多路传输的二甲基化 以亮氨酸(DILEU)为基础的生物素辅助MS定量策略 生物样品中瓜氨酸蛋白的表征和相对定量。具体目标3--至 新开发的多面体方法在脑脊液样品瓜氨酸组分析中的应用 来自年龄匹配的无症状认知功能正常成人、临床前阶段3的个人和轻度 认知障碍(MCI)和阿尔茨海默病。总之，我们提出的实验将开发一种新的基于MS的 能够大规模相对定量复杂生物样品中的瓜氨酸蛋白的方法。 这也是第一次研究瓜氨酸在AD中的作用，促进了我们对AD和 为发现新的AD生物标志物提供了机会。

ABSTRACT Protein citrullination as an enzymatic post-translational modification (PTM) plays important roles in the onset and progression of many diseases, including rheumatoid arthritis, neurodegenerative diseases, diabetes and cardiovascular problems. Current citrullination studies have been primarily relying on immunohistochemistry and Western blotting techniques, and suffer from lack of effective methods for qualitative and quantitative analysis of citrullinated proteins in clinically relevant samples, limiting our understanding of this disease-related PTM. Mass spectrometry (MS) has become a powerful tool for proteomics and site-specific analysis of many PTMs including acetylation, phosphorylation and glycosylation. However, MS-based citrullination analysis presents significant challenge due to the lack of effective methods for specific enrichment of citrullinated proteins in biological samples, and tandem-MS based approaches enabling accurate identification of citrullinated peptides and reliable assignment of citrullination site. The primary goal of this proposal is to develop a multi-faceted approach integrating biotin tag-assisted enrichment method, isobaric tag-based quantitative strategy with MS- based technologies for simultaneous enrichment, large-scale characterization and relative quantitation of citrullinated proteins in biological samples, and apply this multi-faceted approach to investigate the roles of citrullination in Alzheimer’s disease (AD). We propose the following specific aims: Specific Aim 1 – To develop a novel biotin tag-assisted MS-based method for large-scale characterization of citrullinated proteins in biological samples. Specific Aim 2 – To establish a multi-faceted method integrating multiplexed dimethylated leucine (DiLeu)-based quantitation strategy with biotin-assisted MS-based method for simultaneous characterization and relative quantitation of citrullinated proteins from biological samples. Specific Aim 3 – To apply the newly-developed multi-faceted method for citrullinome analysis of cerebrospinal fluid (CSF) samples from age-matched asymptomatic cognitively-healthy adults, preclinical stage 3 individuals and patients with mild cognitive impairment (MCI) and AD. Collectively, our proposed experiments will develop a novel MS-based method enabling large-scale and relative quantitation of citrullinated proteins in complicated biological samples. This is also the first time that the roles of citrullination in AD will be studied, advancing our knowledge of AD and providing an opportunity for new AD biomarker discovery.