Cochlear Nucleus Proteomics in a Mouse Model of Age-Related Hearing Loss

描述（由申请人提供）：患有阿勒的人的中枢听觉通路会发生变化。我们的研究（以及其他一些最近的研究）表明，耳蜗核中参与处理和传输用于声音定位和言语感知某些方面的定时信息的关键“网关”细胞改变了耳聋动物的突触功能和电兴奋性。这些适应性变化是由于特定蛋白质（受体和离子通道）以及其他尚未鉴定的蛋白质表达的变化。确定潜在的蛋白质变化将有助于了解大脑如何对耳聋做出反应，并应有助于设计刺激方案，以及可能为未来的基因治疗提供信息，优化中枢听觉通路的刺激和康复。该建议的重点是表征年龄相关性听力损失（阿勒）小鼠模型中耳蜗前腹侧核（AVCN）的蛋白质组学变化。虽然许多研究都集中在耳蜗（主要听觉器官）的缺陷上，但对听觉脑干中的蛋白质组变化知之甚少。该方案包括两个靶向蛋白质组学方法。我们的第一个目标是表征一种膜蛋白，根据我们的电生理数据在阿勒进展过程中发生变化-离子型谷氨酸受体2。由于已知这种蛋白质的异构体变异和翻译后修饰会影响其活性，因此我们将在基于凝胶和基于亲和力的蛋白质纯化后，采用自上而下和自下而上的质谱分析来研究这种蛋白质。这种蛋白质的深入表征应该表明阿勒中被破坏的途径。我们的第二个目标是鉴定在阿勒进展过程中发生变化的任何膜蛋白。这将通过使用传统的亚细胞分级分离结合比较性1D-凝胶电泳和新型的基于FTICR的质谱法进行差异蛋白质表达分析来实现。使用与FTICR-MS偶联的高分辨率多维液相色谱法，将来自正常和阿勒小鼠的分级分离的膜蛋白的肽用乙酸酐或O3-乙酸酐乙酰化，并组合。这些肽双联体的比率指示相对蛋白质表达水平，而肽质量和MS/MS裂解模式识别蛋白质。

DESCRIPTION (provided by applicant): People that have had AHL will have changes in their central auditory pathways. Our studies (and some other recent studies) have shown that the critical "gateway" cells in the cochlear nucleus that are involved in processing and transmitting timing information used for sound localization and some aspects of speech perception have altered synaptic function and electrical excitability in deaf animals. These adaptations result from changes in the expression of specific proteins (receptors and ion channels), as well as other proteins that have not yet been identified. Identifying the underlying protein changes will help to understand how the brain responds to deafness, and should help to design stimulus protocols, as well as possibly inform future gene therapies, that optimize the stimulation and rehabilitation of the central auditory pathway. The focus of this proposal is to characterize the proteomic changes in the anterior ventral cochlear nucleus (AVCN) in a murine model of age-related hearing loss (AHL). While many studies have focused on defects in the cochleus, the primary auditory organ, less is known about the proteomic changes in the auditory brainstem. This proposal consists of a two targeted-proteomic approaches. Our first aim is to characterize a membrane protein that changed during AHL progression based on our electrophysiological data -- the ionotropic glutamate receptor 2. Since isoform variation and posttranslational modification of this protein is known to affect its activity, we will investigate this protein with top-down and bottom-up mass spectrometric analysis after gel-based and affinity-based protein purification. An in-depth characterization of this protein should indicate pathways which are disrupted in AHL. Our second aim is identification of any membrane proteins that change during AHL progression. This will be accomplished by using traditional subcellular fractionation combined with comparative 1 D-gel electrophoresis and and novel FTICR-based mass spectrometry-based approaches for differential protein expression analysis. Using high-resolution multi-dimensional liquid chromatography coupled to FTICR-MS, peptides from fractionated membrane proteins from normal and AHL mice are acetylated with either acetic anhydride or 03-acetic anhydride, and combined. The ratios of these peptide doublets indicate relative protein expression levels, while the peptide masses and MS/MS fragmentation patterns identify the protein.