Defining the IsoAspartome

定义 IsoAspartome

• 批准号: 7879305
• 负责人: Cheng Lin
• 金额: $ 29.62万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7879305
• 关键词: Anthrax Vaccines; Antibodies; Antigens; Asparagine; Aspartic Acid; Bacillus anthracis; Biological Products; Cardiovascular Diseases; Cardiovascular system; Cells; Data; Dependence; Diagnostic; Disease; Dissociation; Electrons; Enzymes; Erythrocyte Aging; Fourier transform ion cyclotron resonance; In Vitro; Individual; Industry; Isoaspartic Acid; Kinetics; Methods; Modification; Monoclonal Antibodies; Oxidative Stress; Peptides; Phase; Post-Translational Protein Processing; Principal Investigator; Proteins; Proteomics; Reaction; Recombinants; Research; Side; Site; Specificity; Structure; Superoxide Dismutase; System; Technology; Temperature; Testing; Theoretical model; Therapeutic; Thermodynamics; Time; Transferase; Update; Vaccines; Vertebral column; base; carbene; deamidation; interest; mass spectrometer; neoplastic cell; new technology; repair enzyme; repaired; research study; synthetic peptide; theories

DESCRIPTION (provided by applicant): Formation of aspartic acid and isoaspartic acid by spontaneous, nonenzymatic degradation of asparagine is THE most common post translational modification in proteins. While deamidation rates have been studied extensively and correlated with many diseases, their breakdown products have not. Isoaspartic acid is generally considered to be worse than aspartic acid since it is generally more destabilizing due to the extra methylene group in the protein backbone chain. Asp/isoAsp formation rates have not been studied due to the difficulty of distinguishing them, but we have recently developed new technology for doing this that will make this research accessible. This project, therefore, has the basic aim of applying our new technology to determining rates of formation of Asp and isoAsp in proteins first on a synthetic peptide level, then on a clean protein level, and finally on a whole-cell proteomics level. This technology involves using Electron Capture Dissociation (ECD) on FTICR mass spectrometers to detect diagnostic marker peaks, c+57, Z-57, and M-60 (see preliminary data) and plot them as a function of time, primary and secondary sequence structure, pH, and temperature thus studying their kinetics and thermodynamics of formation in relevant reaction phase-space. We have already shown that this can be done with individual peptides and proteins, and we expect it to be possible on a proteomics scale using LC/MS/MS experiments where ECD is used as the fragmentation mechanism. The resulting experimental data will be used to update and calibrate theoretical models of deamidation rates to include rates of formation of Asp and isoAsp. A secondary aim is to study the structural dependence of the PIMT repair enzyme (which converts isoAsp to Asp). This enzyme has been used extensively to detect the presence of isoAsp in a wide variety of systems from in vitro peptides to whole tumor cells to studies of erythrocyte aging. Finally, this study will apply these methods to detailed study of isoaspartic acid formation rates via both deamidation and aspartic acid isomerization in bacillus anthracis vaccine protective antigen, monoclonal antibodies used as biopharmaceuticals, and in proteins of interest in cardiovascular disease.

性状（由申请人提供）：天冬酰胺自发非酶降解形成天冬氨酸和异天冬氨酸是蛋白质中最常见的翻译后修饰。虽然脱酰胺率已被广泛研究，并与许多疾病相关，但其分解产物却没有。异天冬氨酸通常被认为比天冬氨酸更差，因为它通常由于蛋白质主链中的额外亚甲基而更不稳定。由于难以区分它们，尚未研究Asp/isoAsp的形成率，但我们最近开发了新的技术，可以进行这项研究。因此，该项目的基本目标是应用我们的新技术，首先在合成肽水平上，然后在清洁蛋白质水平上，最后在全细胞蛋白质组学水平上确定蛋白质中Asp和isoAsp的形成速率。该技术涉及在FTICR质谱仪上使用电子捕获解离（ECD）来检测诊断标记物峰c+57、Z-57和M-60（参见初步数据），并将它们绘制为时间、一级和二级序列结构、pH和温度的函数，从而研究它们在相关反应相空间中形成的动力学和热力学。我们已经表明，这可以用单个肽和蛋白质来完成，并且我们预计使用LC/MS/MS实验在蛋白质组学规模上是可能的，其中ECD用作片段化机制。所得实验数据将用于更新和校准脱酰胺速率的理论模型，以包括Asp和isoAsp的形成速率。第二个目的是研究PIMT修复酶（将isoAsp转化为Asp）的结构依赖性。这种酶已广泛用于检测各种系统中isoAsp的存在，从体外肽到整个肿瘤细胞再到红细胞老化研究。最后，本研究将应用这些方法详细研究炭疽杆菌疫苗保护性抗原、用作生物药物的单克隆抗体和心血管疾病相关蛋白质中通过脱酰胺和天冬氨酸异构化的异天冬氨酸形成率。