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

一种新的多方面方法，用于生物样品中瓜氨酸蛋白的大规模表征和相对定量及其在阿尔茨海默病中的应用

• 批准号: 9763403
• 负责人: LINGJUN LI
• 金额: $ 18.49万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-15 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9763403
• 关键词: Acetylation; Adult; Age; Alzheimer' s Disease; Alzheimer’s disease biomarker; Antibodies; Arginine; Biological; Biotin; Biotinylation; Cardiovascular Diseases; Cardiovascular system; Cell Extracts; Cerebrospinal Fluid; Charge; Chemicals; Citrulline; Cognitive; Communities; Diabetes Mellitus; Disease; Disease Pathway; Disease Progression; Dissociation; Electron Transport; Enzymes; Family; Glial Fibrillary Acidic Protein; Goals; Hela Cells; Hippocampus (Brain); Homeostasis; Hybrids; Immune response; Immune system; Immunofluorescence Immunologic; Immunohistochemistry; Immunologic Techniques; Ions; Knowledge; Label; Leucine; Mass Spectrum Analysis; Methods; Modification; Neurodegenerative Disorders; Onset of illness; Pathogenicity; Patients; Peptides; Phosphorylation; Physiological; Play; Post-Translational Protein Processing; Preparation; Property; Protein Conformation; Protein-arginine deiminase; Proteins; Proteomics; Qualitative Methods; Reporter; Research; Resolution; Rheumatoid Arthritis; Role; Sampling; Site; Streptavidin; Techniques; Technology; Time; Tissues; To autoantigen; Vimentin; Western Blotting; base; biomarker discovery; citrullinated protein; clinically relevant; comparative; differential expression; experimental study; glycosylation; individual patient; intermolecular interaction; method development; mild cognitive impairment; novel; pre-clinical; protein folding; protein structure function; tandem mass spectrometry; tool

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.

抽象的 蛋白质瓜氨酸化作为一种​​酶促翻译后修饰 (PTM) 在发生和发展过程中发挥着重要作用。 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 目前的瓜氨酸化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 临床相关样品中的瓜氨酸蛋白，限制了我们对这种疾病相关 PTM 的理解。 光谱测定 (MS) 已成为许多 PTM 的蛋白质组学和位点特异性分析的强大工具，包括 然而，基于 MS 的瓜氨酸化分析显示出显着的结果。 由于缺乏有效的方法来特异性富集生物中的瓜氨酸蛋白，因此面临挑战 样品和基于串联质谱的方法能够准确鉴定瓜氨酸肽并可靠 该提案的主要目标是开发一种多方面的方法。 将生物素标签辅助富集方法、基于同量异位标签的定量策略与 MS 相结合 基于同步富集、大规模表征和相对定量的技术 生物样品中的瓜氨酸蛋白，并应用这种多方面的方法来研究 我们提出以下具体目标：具体目标 1 – 开发一种基于生物素标签辅助 MS 的新型方法，用于大规模表征瓜氨酸蛋白 具体目标 2 – 建立一种整合多重二甲基化的多方面方法。 基于亮氨酸 (DiLeu) 的定量策略与基于生物素辅助 MS 的方法同时进行 生物样品中瓜氨酸蛋白的表征和相对定量。 具体目标 3 – 目标 应用新开发的多方面方法对脑脊液 (CSF) 样品进行瓜氨酸组分析 来自年龄匹配的无症状认知健康成年人、临床前 3 期个体和轻度患者 总的来说，我们提出的实验将开发一种新型的基于 MS 的方法。 该方法能够对复杂生物样品中的瓜氨酸蛋白进行大规模相对定量。 这也是首次研究瓜氨酸在 AD 中的作用，推进了我们对 AD 和 AD 的认识。 为新的 AD 生物标志物发现提供了机会。