Proteome-Wide Characterization of Phosphoproteins

磷蛋白的全蛋白质组表征

• 批准号: 6801415
• 负责人: RICHARD S MORRISON
• 金额: $ 18万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6801415
• 关键词: deuterium; gas chromatography mass spectrometry; high performance liquid chromatography; infrared spectrometry; interferometry; ion cyclotron resonance spectrometry; laboratory mouse; method development; neurons; p53 gene /protein; phosphopeptides; phosphoproteins; phosphorylation; posttranslational modifications; protein quantitation /detection; protein structure function; tissue /cell culture

DESCRIPTION (provided by applicant): Due to the importance of reversible phosphorylation in virtually all aspects of cell function and development, there exists a need to develop better methods to identify and quantify changes in the phosphorylation states of proteins on a proteome-wide level. In this R21 project, we will develop and apply new approaches, termed phosphopeptide isotope coded affinity tags (PhIAT), for obtaining proteome-wide identification and precise measurements of differences in the phosphorylation states of the proteins extracted from p53+/+ mouse cortical neurons. Our approach will utilize proteome-wide stable isotope and biotin labeling of phosphopeptides to enable high affinity isolation of phosphopeptides. We will use data-dependent tandem mass spectrometry (MS/MS) and Fourier transform ion cyclotron resonance mass spectrometry (FTICR/MS) to identify phosphorylated peptides that can function as accurate phosphopeptide mass tags (APMTs) to uniquely identify phosphorylated proteins. The approach will provide for high affinity isolation of phosphopeptides, be at least 3 orders of magnitude more sensitive than existing 2-D PAGE methodologies, and be able to rapidly identify and measure relative phosphorylation states for thousands of proteins in a single analysis. We will apply this technology to quantify differences in the relative phosphorylation state of proteins from p53+/+ and p53-/- cortical neurons treated with an apoptotic stimulus. The later phase of this project will develop methods that concomitantly combine PhIAT and ICAT labeling to identify proteins in glutamate or camptothecin treated p53+/+ cortical neurons that undergo changes in either their phosphorylation state or relative abundance compared to non-treated cells. By combining the PhIAT and ICAT strategies on treated p53+/+ neurons, we will be able to identify proteins that undergo a change in phosphorylation without a corresponding change in expression, or vice versa. The development of this capability will ultimately provide the broadest present proteome coverage since changes in protein abundance, as well as changes in protein phosphorylation states will be identifiable in a single experiment.

描述（由申请人提供）：由于可逆性的重要性， 磷酸化在细胞功能和发育的几乎所有方面， 需要开发更好的方法来确定和量化变化 在蛋白质组水平上的磷酸化状态。在R21中 项目，我们将开发和应用新的方法，称为磷酸肽 同位素编码亲和标签（PhIAT），用于获得蛋白质组范围的鉴定 和精确测量磷酸化状态的差异， 从p53+/+小鼠皮层神经元提取的蛋白质。我们的方法将 利用磷酸肽的蛋白质组范围的稳定同位素和生物素标记， 能够高亲和性分离磷酸肽。我们将使用数据依赖 串联质谱和傅里叶变换离子回旋共振 质谱（FTICR/MS），以鉴定磷酸化肽， 作为准确的磷酸肽质量标签（APMT）来唯一识别 磷酸化蛋白质该方法将提供高亲和力分离 磷酸肽，至少3个数量级以上的敏感性比 现有的2-D PAGE方法，并能够快速识别和测量 在一次分析中就能检测出数千种蛋白质的相对磷酸化状态。 我们将应用这项技术来量化相对 来自p53+/+和p53-/-皮质神经元的蛋白质的磷酸化状态 用凋亡刺激物处理。该项目的后期将 开发同时结合联合收割机PhIAT和ICAT标记的方法， 谷氨酸或喜树碱处理的p53+/+皮质神经元中的蛋白质， 在磷酸化状态或相对丰度上发生变化 与未处理的细胞相比。通过结合PhIAT和ICAT战略， 处理p53+/+神经元，我们将能够识别蛋白质， 磷酸化的改变而没有相应的表达改变，或 亦然这种能力的发展将最终提供最广泛的 由于蛋白质丰度的变化， 蛋白质磷酸化状态的变化将在单一的 实验