Proteome-Wide Characterization of Phosphoproteins

描述(申请人提供)：由于可逆性的重要性 在细胞功能和发育的几乎所有方面都有磷酸化， 需要开发更好的方法来识别和量化变化 在蛋白质组水平上处于蛋白质的磷酸化状态。在此R21中 项目，我们将开发和应用新的方法，称为磷肽 同位素编码亲和标签(PhIAT)，用于获得蛋白质组范围的鉴定 和精确测量不同磷酸化状态的差异 从P53+/+小鼠皮质神经元中提取的蛋白质。我们的方法将 利用蛋白质组稳定同位素和生物素标记磷酸肽 实现对磷酸肽的高亲和力分离。我们将使用数据依赖 串联质谱仪与傅里叶变换离子回旋共振 质谱学(FTICR/MS)鉴定可以 用作准确的磷肽质量标签(APMT)，以唯一识别 磷酸化的蛋白质。该方法将提供高亲和力分离 在磷酸肽中，敏感度至少比 现有的2-D PAGE方法，并能够快速识别和测量 在一次分析中，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 P53+/+和P53-/-皮质神经元蛋白质的磷酸化状态 用细胞凋亡性刺激进行治疗。该项目的后期阶段将 开发同时结合PHIAT和ICAT标记的方法来识别 谷氨酸或喜树碱处理的P53+/+皮质神经元中的蛋白质 经历它们的磷酸化状态或相对丰度的变化 与未处理的细胞相比。通过将PhIAT和ICAT战略结合在一起 经过处理的p53+/+神经元，我们将能够识别经历了 在没有相应表达变化的情况下磷酸化的变化，或者相反 反过来说。这种能力的发展最终将提供最广泛的 目前的蛋白质组覆盖范围自蛋白质丰度变化以来，以及 蛋白质磷酸化状态的变化将在单个 做实验。

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.