PROTEOMICS

蛋白质组学

• 批准号: 7568471
• 负责人: John R Yates III
• 金额: $ 41.24万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7568471
• 关键词: Age of Onset; Aging; Amyloid beta-Protein; Area; Biochemical; Biological Assay; Data; Disease; Dissociation; Drug Metabolic Detoxication; Electron Transport; Hybrids; Laboratories; Link; Methods; Molecular; Nerve Degeneration; Post-Translational Protein Processing; Process; Production; Proteins; Proteolysis; Proteomics; RNA Interference; Resolution; Scanning; System; Techniques; Technology; abstracting; experience; instrument; mass spectrometer; polypeptide; protein aggregate; research study; transcription factor

Abstract The central hypothesis of this proposal is that continued production of aggregation prone proteins leads to age onset proteotoxicity and eventually disease. Two areas will be studied using proteomics technology. The first is to identify the proteins involved in disassembly/proteolysis of protein aggregates. By using newly developed assays in the Kelly laboratory, disassembly and proteolysis activities will be followed over the course of biochemical enrichment. Proteomics will be used during the enrichment process to follow and assess the course of enrichment and then finally to identify the proteins responsible for the activity. Detoxification activities are under the control of the transcription factors HSF-1 and DAF-16. We will identify the proteins whose expression levels are under the control of these transcription factors by using quantitative proteomics when the levels of HSF-1 and DAF-16 are reduced by RNAi. The methods, techniques, and experience are available in the proteomics core to conduct these analyses. The core will employ large-scale protein identification technology developed in our lab called MudPIT (Multi-dimensional Protein Identification Technology). To perform MudPIT experiments we have several different types of mass spectrometers that can be used including LTQ-Orbitrap, LTQ and LTQ-ETD (Electron Transfer Dissociation) systems. Each instrument has particular strengths that can be applied to these projects. The LTQ-Orbitrap produces high resolution and high mass accuracy data that is good for confident protein identifications, the discovery of post translational modifications, and accurate quantitation. The LTQ is a slightly more sensitive and faster scanning instrument than the hybrid and can be used when sensitivity and sequence coverage are important. The LTQ-ETD system uses a new dissociation technique that is good for fragmenting large polypeptides. This particular instrument may have an advantage when attempting to identify proteins from aggregates or proteins that do not digest well.

抽象的 该提议的核心假设是，持续产生易于聚集的蛋白质导致 年龄发作蛋白毒性，最终疾病。将使用蛋白质组学技术研究两个领域。 首先是鉴定蛋白质聚集体拆解/蛋白水解涉及的蛋白质。通过新的 在凯利实验室，拆卸和蛋白水解活动中开发的测定法会遵循 生化富集的过程。蛋白质组学将在富集过程中用于遵循和 评估富集的过程，然后最终确定负责活动的蛋白质。 排毒活性在转录因子HSF-1和DAF-16的控制之下。我们将确定 表达水平在这些转录因子控制下的蛋白质通过使用 当RNAi降低HSF-1和DAF-16的水平时，定量蛋白质组学。方法， 蛋白质组学核心可以使用技术和经验来进行这些分析。核心意愿 在我们的实验室中采用大规模蛋白质识别技术，称为Mudpit（多维 蛋白质识别技术）。要执行泥坑实验，我们有几种不同类型的质量 可以使用的光谱仪，包括LTQ-Orbitrap，LTQ和LTQ-ETD（电子传递解离） 系统。每种仪器都有可以应用于这些项目的特殊优势。 LTQ-Orbitrap 产生高分辨率和高质量精度数据，这对自信蛋白质识别有利， 发现后翻译修改和准确的定量。 LTQ更敏感 和更快的扫描仪器比混合动力车，可以在灵敏度和序列覆盖率时使用 很重要。 LTQ-ETD系统使用一种新的解离技术，适合分裂 大多肽。尝试识别蛋白质时，该特定仪器可能会有优势 来自骨料或蛋白质的消化不佳。