Using stable isotope labeling/mass spectrometry to probe Cdk1 multisite phosphory

使用稳定同位素标记/质谱法探测 Cdk1 多位点磷酸

• 批准号: 8019007
• 负责人: Emmanuel Joel Chang
• 金额: $ 16.02万
• 依托单位: YORK COLLEGE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8019007
• 关键词: Address; Adenosine Triphosphate; Alzheimer' s Disease; Binding; Biochemistry; Biological; Biological Assay; CDC2 Protein Kinase; Cell Cycle; Cell Proliferation Regulation; Cell division; Cells; Characteristics; Collaborations; Complex; Coupled; Cyclin-Dependent Kinases; Cyclins; Dependence; Development; Dissection; Embryonic Development; Enzymes; Eukaryota; Family; Funding; Future; Glycogen Synthase Kinases; Goals; Health; Human; Human Biology; In Vitro; Inflammation; Kinetics; Knowledge; Label; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Mediating; Mentors; Metabolic; Methods; Mission; Modeling; Mutate; Nature; Peptides; Phosphorylation; Phosphorylation Site; Phosphotransferases; Pilot Projects; Population; Process; Property; Protein Dynamics; Proteins; Proteomics; Publications; Publishing; Reaction; Reagent; Regulation; Research; Research Personnel; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Saccharomycetales; Site; Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stable Isotope Labeling; System; Techniques; Technology; Temperature; Time; United States National Institutes of Health; Variant; Work; Yeasts; base; casein kinase; dissemination research; in vitro Assay; in vivo; innovation; inorganic phosphate; insight; mutant; nervous system disorder; protein complex; skills

DESCRIPTION (provided by applicant): The proposed pilot project intends to develop a quantitative mass spectrometric method for studying multisite phosphorylation in a detailed and controlled manner, and to apply that method to the multisite phosphorylation of cyclin-dependent kinase substrates. The detailed study of multisite phosphorylation has been hampered by the lack of techniques capable of quantitatively analyzing phosphorylation dynamics under well-defined conditions. Here, stable isotope-labeling coupled to mass spectrometry will be applied to in vitro phosphorylation reactions between the budding yeast cyclin-dependent kinase Cdk1 and a number of substrate molecules. Stable isotope-labeling/mass spectrometry is a well-established technique with the appropriate sensitivity, dynamic range and precision for making the measurements that will be required to accurately quantify protein phosphorylation in a time-resolved, site-specific manner. Published and unpublished preliminary studies have been performed to show that Cdk1 can be isolated from yeast cells in an active form, and can be used for in vitro phosphorylation reactions. This proposal aims to understand multisite Cdk1 phosphorylation as a biophysical system, and therefore requires the precise control afforded by in vitro kinetic studies. The in vitro approach is preferred because hidden variables are invariably present in the complicated in vivo cellular milieu, such as cross-talk between related enzymes and the presence of diverse protein populations within a single culture. Specifically, this proposal aims to determine the processivity or distributivity of Cdk1 phosphorylation; and the order and dependence, if any, of sites are phosphorylated. Furthermore, Cdk1 has 9 potential in vivo cyclin binding partner/activators present at different points in cell cycle that have partially overlapping specificity and function. Results of these multisite phosphorylation studies may shed insight into differences between the cyclins and modes of cyclin specificity. A major part of the pilot project will also be the professional development of the PI through collaboration with a highly successful mentor, research dissemination, establishment of collaborations, and ultimately, the acquisition of future funding. RELEVANCE TO PUBLIC HEALTH: This proposal aims to utilize leading-edge analytical technology to understand the activity of cyclin dependent kinases, a main player in cell division. Knowledge gained from the proposed study on the model eukaryote S. cerevisiae will have value in elucidating principles that also human biology, particularly as it relates to regulation of cell proliferation and cancer. The innovative in vitro phosphorylation/quantitative mass spectrometry approach proposed also lends itself readily to studying other multisite enzyme systems, such as the glycogen synthase kinases, and casein kinases-proteins that are intimately involved in processes such as metabolic regulation, inflammation, and Alzheimer's disease and other neurological disorders.

描述（由申请人提供）：拟议的试点项目旨在开发一种定量质谱方法，以详细和受控的方式研究多位点磷酸化，并将该方法应用于细胞周期蛋白依赖性激酶底物的多位点磷酸化。多位点磷酸化的详细研究一直受到阻碍，缺乏技术能够定量分析磷酸化动力学条件明确。在这里，稳定的同位素标记耦合质谱将适用于在体外的芽殖酵母细胞周期蛋白依赖性激酶Cdk 1和底物分子之间的磷酸化反应。稳定同位素标记/质谱法是一种成熟的技术，具有适当的灵敏度，动态范围和精度，用于以时间分辨，位点特异性方式准确定量蛋白质磷酸化所需的测量。已经进行了已发表和未发表的初步研究，以表明Cdk 1可以以活性形式从酵母细胞中分离，并可用于体外磷酸化反应。该建议旨在了解多位点Cdk 1磷酸化作为一个生物物理系统，因此需要在体外动力学研究提供的精确控制。体外方法是优选的，因为隐藏变量总是存在于复杂的体内细胞环境中，例如相关酶之间的串扰和单一培养物中不同蛋白质群体的存在。具体而言，该建议旨在确定Cdk 1磷酸化的持续合成性或分配性;以及磷酸化位点的顺序和依赖性（如果有的话）。此外，Cdk 1有9个潜在的体内细胞周期蛋白结合伴侣/激活剂存在于细胞周期的不同点，具有部分重叠的特异性和功能。这些多位点磷酸化研究的结果可能会揭示细胞周期蛋白和细胞周期蛋白特异性模式之间的差异。试点项目的一个主要部分也将是PI的专业发展，通过与一个非常成功的导师合作，研究传播，建立合作，并最终获得未来的资金。与公共卫生的关系：该提案旨在利用领先的分析技术来了解细胞周期蛋白依赖性激酶的活性，细胞分裂的主要参与者。从模式真核生物S.酿酒酵母将在阐明人类生物学原理方面具有价值，特别是因为它涉及细胞增殖和癌症的调节。提出的创新的体外磷酸化/定量质谱方法也很容易用于研究其他多位点酶系统，如糖原合酶激酶和酪蛋白激酶-密切参与代谢调节、炎症和阿尔茨海默病等过程的蛋白质。

项目成果

Studies on quantitative phosphopeptide analysis by matrix-assisted laser desorption/ionization mass spectrometry without label, chromatography or calibration curves.

• DOI: 10.1002/rcm.7063
• 发表时间: 2014-12-30
• 期刊: RAPID COMMUNICATIONS IN MASS SPECTROMETRY
• 影响因子: 2
• 作者: Ho, Hsin-Pin;Rathod, Pratikkumar;Louis, Marissa;Tada, Christine K.;Rahaman, Sherida;Mark, Kevin J.;Leng, Jin;Dana, Dibyendu;Kumar, Sanjai;Lichterfeld, Mathias;Chang, Emmanuel J.
• 通讯作者: Chang, Emmanuel J.