Eukaryotic Post-Translational Modifications

真核翻译后修饰

• 批准号: 7104814
• 负责人: NEIL L KELLEHER
• 金额: $ 28.98万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7104814
• 关键词: Saccharomyces cerevisiae; computer program /software; computer system design /evaluation; electrospray ionization mass spectrometry; eukaryote; fungal proteins; interferometry; mass spectrometry; nanotechnology; posttranslational modifications; protein structure; proteomics; technology /technique development

DESCRIPTION (provided by applicant): We propose to characterize 1000 protein forms present in the yeast, Saccharomyces cerevisiae, with unprecedented molecular detail to detect and characterize unexpected mass discrepancies (e.g., covalent modifications) to proteins. This research will combine a completely sequenced genome with recent advances in Fourier-Transform Mass Spectrometry (FTMS) and Electrospray Ionization to translate raw genomic data into biological knowledge and insight. To date, the direct fragmentation of intact proteins has been demonstrated mainly for protein standards or recombinant material. Existing limitations to analyzing intact proteins efficiently include difficulties with solubilization/fractionation, a size constraint, and a lack of dedicated software. However, we will construct a unique measurement platform using an acid-labile surfactant, nanospray robotics, a quadrupole-FTMS Hybrid Instrument, and custom software - all dedicated to realizing measurement throughput. While focusing heavily on technique and software development, it is the biomedical importance of post-translational modifications (PTMs) that drives this research. Targeted for analysis are the 78 proteins from purified yeast ribosomes and 1000 protein forms fractionated directly from its proteome under aerobic and anerobic conditions. In the yeast proteome database, there exist a total of 893 hypothetical and experimentally verified PTMs. We intend to add to this list and enumerate the microheterogeneity, percent occupancy, and dynamic responses of PTMs detected in this study. The research proposed has a pragmatic aspect (i.e., the characterization of unpredictable gene processing events at high detail), but also seeks to establish the analytical infrastructure to eventually contribute in a unique fashion to the Human Proteome Project. The robust detection of eukaryotic PTMs (and quantitation of their changes) is critical to realize the potential of genomic sciences for detecting and fundamentally understanding a broad spectrum of human disease.

描述（由申请人提供）：我们建议以前所未有的分子细节来表征酿酒酵母中存在的 1000 种蛋白质形式，以检测和表征蛋白质的意外质量差异（例如共价修饰）。这项研究将完整测序的基因组与傅里叶变换质谱（FTMS）和电喷雾电离的最新进展相结合，将原始基因组数据转化为生物学知识和见解。迄今为止，完整蛋白质的直接片段化主要针对蛋白质标准品或重组材料得到证实。有效分析完整蛋白质的现有限制包括溶解/分级困难、尺寸限制以及缺乏专用软件。然而，我们将使用酸不稳定表面活性剂、纳米喷雾机器人、四极杆-FTMS 混合仪器和定制软件构建一个独特的测量平台 - 所有这些都致力于实现测量吞吐量。虽然重点关注技术和软件开发，但翻译后修饰 (PTM) 的生物医学重要性推动了这项研究。 分析的目标是来自纯化酵母核糖体的 78 种蛋白质以及在有氧和无氧条件下直接从其蛋白质组中分离出来的 1000 种蛋白质形式。在酵母蛋白质组数据库中，总共存在 893 个假设和实验验证的 PTM。我们打算添加到这个列表中，并列举本研究中检测到的 PTM 的微观异质性、占有率百分比和动态响应。拟议的研究具有务实的一面（即，高度详细地描述不可预测的基因处理事件），但也寻求建立分析基础设施，最终以独特的方式为人类蛋白质组计划做出贡献。真核 PTM 的可靠检测（及其变化的定量）对于实现基因组科学检测和从根本上理解广泛的人类疾病的潜力至关重要。