ATP-BINDING PROTEIN QUANTITATIVE ANALYSIS BY TANDEM MASS SPECTROMETRY

通过串联质谱法对 ATP 结合蛋白进行定量分析

• 批准号: 7420648
• 负责人: Michael MacCoss
• 金额: $ 1.66万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-20 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7420648
• 关键词: DNA repair; binding proteins; family; gene mutation; mass spectrometry; protein binding; proteins; quality of life

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. DNA damage can be deleterious to cell survival and cells have adopted multiple strategies to bypass or physically remove certain specific types of DNA lesions. Also, cells can slow down their progression through different cell cycle phases to provide time for repair to occur and prevent mutation mutations from being propagated. Sequential phosphorylation event plays a key role in activating the DNA repair machinery. In most of the cases, individual signaling network for DNA repair has been well characterized, still little is known about the global regulation of kinase activities and other ATP-binding proteins involved in the process. Heterogeneity and low abundance make the analysis difficult. With an ATP-binding protein enrichment tool, we could selectively enrich a major portion of the kinases and other ATP-binding proteins in budding yeast S. cerevisiae. Combining kinase enrichment tool, peptide chromatography and tandem mass spectrometry, we are able to quantitatively assess changes in kinase expression level. The method developed could well be applied to higher organisms. Small kinase inhibitor molecules are frequently used to dissect signaling pathways or to counteract oncogenic events. However, a functional compensation is established by a homolog kinase of the same family. The phenomenon is true to many examples with serine/threonine kinase families. A global view of kinase expression level at certain cell event would help a better understanding of kinase regulation and drug design.

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。DNA损伤可能对细胞生存有害，细胞已采取多种策略绕过或物理移除某些特定类型的DNA损伤。此外，细胞可以在不同的细胞周期阶段减缓它们的进程，为修复提供时间，并防止突变的传播。序列磷酸化事件在激活DNA修复机制中起着关键作用。在大多数情况下，DNA修复的单个信号网络已经被很好地描述，但对参与这一过程的激酶活性和其他ATP结合蛋白的全球调节知之甚少。非均质性和低丰度给分析带来了困难。利用一种ATP结合蛋白浓缩工具，我们可以选择性地在萌芽酵母中富集大部分的激酶和其他ATP结合蛋白。结合激酶浓缩工具、多肽层析和串联质谱仪，我们能够定量评估激酶表达水平的变化。所开发的方法可以很好地应用于高等生物。小的激酶抑制分子经常被用来剖析信号通路或对抗致癌事件。然而，功能补偿是由同一家族的同源激酶建立的。这种现象在许多丝氨酸/苏氨酸激酶家族的例子中是真实的。对某些细胞事件中的激酶表达水平的全球观察将有助于更好地理解激酶的调节和药物设计。