Time Resolved Assay of Synaptic Enzyme Activity by Mass Spectrometry

通过质谱法对突触酶活性进行时间分辨分析

• 批准号: 8454531
• 负责人: MARY B KENNEDY
• 金额: $ 40.74万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-19 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8454531
• 关键词: Alzheimer' s Disease; Axon; Basic Science; Bathing; Biochemical; Biochemical Pathway; Biological; Biological Assay; Biological Neural Networks; Brain; Cations; Chromatography; Collaborations; Detection; Development; Disease; Electronics; Enzyme Activation; Enzymes; Ethane; Excitatory Postsynaptic Potentials; Excitatory Synapse; Fractionation; Freezing; Health; Hippocampus (Brain); Individual; Information Storage; Ions; Isoelectric Focusing; Isotope Labeling; Laboratories; Liquid substance; Mass Spectrum Analysis; Measurement; Measures; Mental disorders; Methods; Modification; Monitor; Pathway interactions; Peptides; Perfusion; Pharmaceutical Preparations; Phosphopeptides; Physics; Positioning Attribute; Post-Translational Protein Processing; Preclinical Drug Evaluation; Preparation; Process; Propane; Protein Kinase; Proteins; Proteome; Research Support; Resolution; Sampling; Site; Slice; Stimulus; Synapses; Synaptic plasticity; Technology; Time; Tissues; Training; United States National Institutes of Health; brain tissue; design; enzyme activity; high throughput screening; improved; inorganic phosphate; instrument; millisecond; multiple reaction monitoring; protein structure; relating to nervous system; research study; response; scale up; screening; time interval

DESCRIPTION (provided by applicant): We will develop a new method to measure the time courses of activation of biochemical regulatory networks that control changes in synaptic strength which underlie processing and storage of information in neural networks. The proposed method will permit unprecedented time resolution and will enable measurement of the time courses of activation of at least 20, and eventually as many as 50 to 100 enzymes in brain tissue that has been rapidly frozen at intervals as small as one second following an electrical or pharmacological stimulus. The method will be immediately applicable to basic research on, and target development for, mental illnesses and Alzheimer's disease. Upon scale-up, it will be applicable to screening for drugs to treat these diseases. The method will involve substantial adaptation of two existing technologies: "plunge-freezing" and "Selected/ Multiple Reaction Monitoring" (S/MRM) by mass spectrometry. Once developed, both technologies can be scaled up for medium or high throughput screening. The project has three aims. First, we will develop a plunge freeze apparatus to rapidly freeze slices of hippocampal tissue at accurate time intervals following application of a stimulus to the perfused slice. We will accomplish this by making modifications and additions to a plunge-freeze apparatus now commercially available from Leica (Leica EM GP). We will devise an optimal design for a sample chamber to maintain the health of slices during perfusion, and to deliver electrical stimuli to the Schaffer collateral pathway, a major hippocampal axon tract, prior to rapidly freezing the slice by plunging it into a -1900 C liquid propane/ethane bath. We estimate that freezing time to the center of the slice upon plunge will be ~ 200 msecs or less. This freezing time is compatible with a resolution of one second for time intervals following application of a discrete stimulus. Second, we will develop methods to measure the activation of a panel of 20-25 protein kinases or their key substrate proteins located at positions in the regulatory networks that are believed to control synaptic plasticity in excitatory synapses in the hippocampus. Each enzyme or substrate that we will measure is regulated by addition of a phosphate group to key residues in the protein structure. Mass spectrometry will be used to measure changes in the levels of these phosphorylated sites in the frozen slice tissue. Third, once the assays are developed, we will carry out "proof of principle" experiments by combining the technologies developed in Aims 1 and 2 to acquire time courses of activation of each the enzymes in hippocampal slices after delivery of stimuli that alter synaptic plasticity.

描述（由申请人提供）：我们将开发一种新的方法来测量生化调节网络激活的时间过程，所述生化调节网络控制突触强度的变化，所述突触强度是神经网络中信息处理和存储的基础。所提出的方法将允许前所未有的时间分辨率，并将能够测量至少20个，最终多达50至100个酶的激活在脑组织中的时间过程，已被迅速冻结在一个电或药理刺激后的间隔小至一秒。该方法将立即适用于精神疾病和阿尔茨海默病的基础研究和目标开发。扩大规模后，它将适用于筛选治疗这些疾病的药物。该方法将涉及对两种现有技术的实质性调整：通过质谱法进行的“骤降冷冻”和“选择/多反应监测”（S/MRM）。一旦开发出来，这两种技术都可以扩大规模，用于中等或高通量筛选。 该项目有三个目标。首先，我们将开发一种插入式冷冻装置，在对灌注切片施加刺激后以准确的时间间隔快速冷冻海马组织切片。我们将通过对现在可从Leica（Leica EM GP）商购获得的插入冷冻装置进行修改和添加来实现这一点。我们将设计一个最佳设计的样品室，以保持健康的切片在灌注过程中，并提供电刺激的谢弗侧支通路，一个主要的海马轴突束，然后快速冷冻切片，将其投入到-1900 ℃的液体丙烷/乙烷浴。我们估计，插入时到切片中心的冻结时间约为200毫秒或更短。该冻结时间与施加离散刺激后时间间隔的一秒分辨率兼容。其次，我们将开发方法来测量一组20-25蛋白激酶或其关键底物蛋白的激活，这些蛋白激酶或其关键底物蛋白位于被认为控制海马兴奋性突触中突触可塑性的调节网络中的位置。我们将测量的每种酶或底物都是通过在蛋白质结构中的关键残基上添加磷酸基团来调节的。质谱法将用于测量冷冻切片组织中这些磷酸化位点水平的变化。第三，一旦开发了检测方法，我们将通过结合目标1和2中开发的技术来进行“原理证明”实验，以获得在传递改变突触可塑性的刺激后海马切片中每种酶的激活的时间过程。