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.

摘要 瓜氨酸蛋白作为一种酶促翻译后修饰（PTM）， 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 心血管问题目前的瓜氨酸研究主要依赖于免疫组织化学， 蛋白质印迹技术，缺乏有效的方法进行定性和定量分析， 瓜氨酸化蛋白在临床相关的样本，限制了我们对这种疾病相关的PTM的理解。质量 质谱（MS）已成为蛋白质组学和许多PTM位点特异性分析的有力工具，包括 乙酰化、磷酸化和糖基化。然而，基于MS的瓜氨酸分析显示， 由于缺乏有效的方法来特异性富集瓜氨酸化蛋白， 样品和基于串联质谱的方法，能够准确鉴定瓜氨酸化肽， 瓜氨酸位点的分配。该提案的主要目标是制定一种多方面的方法 将生物素标记辅助富集方法、同量异位素标记定量策略与MS- 同时富集、大规模表征和相对定量的基础技术 瓜氨酸化蛋白的生物样品，并应用这种多方面的方法来研究 瓜氨酸在阿尔茨海默病（AD）中的作用。我们提出以下具体目标：具体目标1- 建立一种新的生物素标记辅助的基于MS的瓜氨酸化蛋白质大规模表征方法 在生物样本中。具体目标2-建立一种多方面的方法， 基于亮氨酸（DiLeu）的定量策略与生物素辅助的基于MS的方法同时用于 来自生物样品的瓜氨酸化蛋白的表征和相对定量。具体目标3-至 将新开发的多方面方法应用于脑脊液（CSF）样本的瓜氨酸组分析 来自年龄匹配的无症状认知健康成人、临床前3期个体和轻度 认知障碍（MCI）和AD。总的来说，我们提出的实验将开发一种新的基于MS的 能够大规模和相对定量复杂生物样品中瓜氨酸化蛋白的方法。 这也是第一次研究瓜氨酸在AD中的作用，推进了我们对AD的认识， 为发现新的AD生物标志物提供了机会。