Defining the IsoAspartome

描述（由申请人提供）：天门冬氨酸自发、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，但其分解产物尚未得到研究。异天冬氨酸通常被认为比天冬氨酸更糟糕，因为它通常更不稳定，因为蛋白质主链中有额外的亚甲基。由于难以区分，Asp/isoAsp的形成速率尚未进行研究，但我们最近开发了一种新技术来进行这项研究，这将使这项研究变得容易。因此，这个项目的基本目标是应用我们的新技术来确定蛋白质中Asp和isoAsp的形成速率，首先在合成肽水平上，然后在清洁蛋白水平上，最后在全细胞蛋白质组学水平上。该技术包括在FTICR质谱仪上使用电子捕获解离（ECD）来检测诊断标记峰c+57、Z-57和M-60（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的形成动力学和热力学。我们已经证明这可以用单个肽和蛋白质来完成，我们希望在使用ECD作为碎片机制的LC/MS/MS实验的蛋白质组学规模上实现这一目标。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。该酶已被广泛用于检测isoAsp在各种系统中的存在，从体外多肽到整个肿瘤细胞，再到红细胞衰老的研究。最后，本研究将把这些方法应用于炭疽芽孢杆菌疫苗保护抗原、生物制药用单克隆抗体和心血管疾病感兴趣的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的详细研究。

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.