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 蛋白柠檬酸作为酶促翻译后修饰（PTM）在发作和 许多疾病的进展，包括类风湿关节炎，神经退行性疾病，糖尿病和 心血管问题。当前的柠檬化研究是主要依赖免疫组织化学和 蛋白质印迹技术，缺乏有效的方法来定性和定量分析 在临床相关样品中，瓜氨酸的蛋白质限制了我们对这种疾病相关PTM的理解。大量的 光谱法（MS）已成为蛋白质组学的强大工具，并对许多PTM的现场分析（包括） 乙酰化，磷酸化和糖基化。但是，基于MS的柠檬硫化分析呈现出重要的 挑战由于缺乏有效的方法来特异性富集瓜氨酸蛋白 样品和基于串联MS的方法可以准确鉴定柠檬油辣椒和可靠的方法 柠檬化地点的分配。该提案的主要目标是开发一种多方面的方法 整合生物素标签辅助富集方法，基于同等标签的定量策略与MS- 基于同时富集，大规模表征和相对定量的技术 生物样品中的瓜氨酸蛋白，并采用这种多方面的方法来研究 柠檬化在阿尔茨海默氏病（AD）中的作用。我们提出以下特定目标：特定目标1 - 开发一种新型生物素标记辅助MS的方法，用于大规模表征瓜氨酸蛋白 在生物样品中。特定目的2 - 建立一种多面方法，整合了多路复用的二甲基化的方法 基于生物素辅助MS的方法的基于亮氨酸（Dileu）的定量策略，用于简单 来自生物样品的瓜氨酸蛋白的表征和相对定量。特定目标3 - 将新开发的多面方法应用用于脑脊液（CSF）样品的瓜氨酸分析 从年龄匹配的无症状认知健康的成年人开始 认知障碍（MCI）和AD。总的来说，我们提出的实验将开发出一种新型基于MS的新型实验 可以在复杂的生物样品中大规模和相对定量的瓜氨酸蛋白。 这也是第一次将柠檬化在广告中的角色进行研究，从而促进我们对广告的了解 为新的AD生物标志物发现提供了机会。