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: 8192670
• 负责人: MARY B KENNEDY
• 金额: $ 47.94万
• 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/8192670
• 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; Screening procedure; 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; 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. PUBLIC HEALTH RELEVANCE: This project will develop a new method for measuring activity in biochemical pathways that do not function properly in individuals with mental illnesses or Alzheimer's disease. The method will be used to support research on the causes of mental illnesses and Alzheimer's disease and for efficient screening for new drugs to treat these diseases.

描述(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进程，该网络控制突触强度的变化，而突触强度是神经网络中信息处理和存储的基础。拟议中的方法将实现前所未有的时间分辨率，并将能够测量脑组织中至少20种、最终多达50到100种酶的激活时间进程，这些酶在电或药物刺激后以小至一秒的间隔快速冻结。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。一旦扩大规模，它将适用于治疗这些疾病的药物筛选。该方法将涉及对现有的两项技术：“骤降冷冻”和“选择性/多反应监测”(S/多反应监测)进行实质性的修改。一旦开发出来，这两项技术都可以扩大到中高通量筛选。该项目有三个目标。首先，我们将开发一种快速冷冻装置，在对灌流的脑片施加刺激后，以准确的时间间隔快速冷冻海马区组织切片。我们将通过对徕卡(徕卡EM GP)现已商业化的俯冲冷冻设备进行修改和增加来实现这一点。我们将为样本室设计一个最佳设计，以在灌流过程中保持切片的健康，并将电刺激传递到Schaffer侧支通路，这是主要的海马轴突束，然后通过将其投入-1900C的丙烷/乙烷液体浴中快速冷冻切片。我们估计，下降到切片中心的冻结时间将在~200毫秒或更短。该冻结时间与施加离散刺激后的时间间隔的1秒分辨率兼容。其次，我们将开发方法来测量一组20-25个蛋白激酶或它们的关键底物蛋白的激活情况，这些蛋白蛋白位于调控网络中被认为控制海马区兴奋性突触突触可塑性的位置。我们将测量的每个酶或底物都是通过在蛋白质结构的关键残基上添加一个磷酸基团来调节的。质谱仪将被用来测量冰冻切片组织中这些磷酸化位点水平的变化。第三，一旦分析方法被开发出来，我们将通过结合目标1和目标2中开发的技术来进行“原则证明”实验，以获得在传递改变突触可塑性的刺激后，海马片中每种酶的激活时间进程。 与公共卫生相关：该项目将开发一种新的方法，用于测量患有精神疾病或阿尔茨海默病的人在生物化学途径中不能正常发挥作用的活动。该方法将用于支持对精神疾病和阿尔茨海默病病因的研究，并用于有效筛选治疗这些疾病的新药。