Time Resolved Assay of Synaptic Enzyme Activity by Mass Spectrometry

描述（由申请人提供）：我们将开发一种新的方法来测量生物化学调节网络激活的时间过程，生物化学调节网络控制突触强度的变化，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种，最终多达50到100种脑组织酶的激活时间过程，这些酶在电刺激或药物刺激后以短至一秒的间隔迅速冻结。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大量改进：“骤降冷冻”和“选择/多重反应监测”（S/MRM）质谱法。一旦开发出来，这两种技术都可以扩大到中通量或高通量筛选。该项目有三个目标。首先，我们将开发一种快速冷冻装置，在对灌注的切片施加刺激后，以准确的时间间隔快速冷冻海马组织切片。我们将通过修改和增加现在从徕卡（徕卡EM GP）市售的暴跌冷冻装置来实现这一目标。我们将设计一种最佳的样品室设计，以保持切片在灌注期间的健康，并在将切片放入-1900℃的液体丙烷/乙烷浴液中快速冷冻之前，向海马主要轴突束Schaffer侧枝通路（Schaffer collateral pathway）传递电刺激。我们估计，在弹射时，到薄片中心的冻结时间为~ 200毫秒或更短。这种冻结时间与应用离散刺激后的时间间隔的一秒分辨率是相容的。其次，我们将开发方法来测量20-25个蛋白激酶或其关键底物蛋白的激活，这些蛋白激酶或底物蛋白位于调节网络中被认为控制海马兴奋性突触突触可塑性的位置。我们将测量的每一种酶或底物都是通过在蛋白质结构的关键残基上添加磷酸基来调节的。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。第三，一旦检测方法被开发出来，我们将结合目标1和目标2中开发的技术进行“原理验证”实验，以获得刺激改变突触可塑性后海马体切片中每种酶的激活时间过程。

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.