Discovery of neuroproteomic determinants of pain transmission using differential and systematic proteome exploration strategies

治疗神经病变动物损伤神经组织后的痛觉过敏和超感痛觉已被广泛研究，但收效甚微。这项研究计划将分三个阶段启动。最初阶段将包括利用原激肽原(PPT-A)基因敲除和野生型小鼠在中枢神经系统中进行差异蛋白质组探索，以鉴定由原速激肽原基因编码的特定蛋白质，重点是速激肽原神经肽的前神经肽。第二阶段将包括中枢神经系统的系统蛋白质组探索，包括神经蛋白质组的鉴定、特征和定量，特别是在经过验证的神经病理性疼痛大鼠模型中，对速激肽神经肽的神经肽进行鉴定、表征和定量。我们发展了从脊髓中分析多肽和蛋白质的专业知识，使用了特定的提取/浓缩技术、消化方法、十二烷基硫酸钠-PAGE、2D凝胶电泳法、毛细管高效液相色谱、离子陷阱质谱仪和MALDI-TOF。将使用多路等压标签技术进行蛋白质定量，以发现生物标记物，以表征蛋白质表达比率图谱，执行主成分分析(PCA)，并定义以速激肽前体基因相关蛋白质为主要焦点的多参数图谱，用于诊断和预后目的。此外，第三个目标将包括评估给予瞬时受体电位通道香草素1(TRPV1)的激动剂/拮抗剂后的神经蛋白质组调节。TRPV1受体参与了疼痛的传递和调制，TRPV1配体在缓解疼痛方面显示出良好的效果。这项建议的总体目标是了解对速激肽前体基因相关蛋白特别感兴趣的神经蛋白的调节机制和条件，并培养更好的机制知识，以支持开发治疗神经病理性和慢性疼痛的新药。

The treatment of pain perception following traumatized nervous tissue leading to hyperalgesia and allodynia in neuropathic animals has been widely investigated with limited success. This research program will be initiated in three phases. The initial phase will include differential proteome exploration in the central nervous system using preprotachykinins (PPT-A) knockout and wild type mice for the identification of specific proteins encoded by the preprotachykinins gene with an emphasis on proneuropeptides of tachykinins neuropeptides. The second phase will include a systematic proteome exploration in the central nervous system including the identification, characterization and quantification of the neuroproteome with a special interest on proneuropeptides of tachykinins neuropeptides in a validated rat model of neuropathic pain. We developed an expertise in the analysis of peptides and proteins from spinal cords using, specific extraction/concentration techniques, digestion methods, SDS-PAGE, 2D gel electropheresis, capillary HPLC, ion trap mass spectrometry and MALDI-TOF. Protein quantification will be performed using multiplexed isobaric tagging techniques for biomarker discovery to characterize protein expression ratio profiles, performed principal component analysis (PCA) and define a multiparametric profile with a primary focus in preprotachykinins gene related proteins for diagnostic and prognostic purposes. Moreover, the third objective will include the evaluation of the neuroproteome regulation following the administration of agonist/antagonist of Transient Receptor Potential Channel Vanilloid 1 (TRPV1). TRPV1 receptors are involved in the transmission and modulation of pain and TRPV1 ligands shown promising results to alleviate pain. The general objective of this proposal is to understand the mechanism and the condition from which neuroproteins with a special interest to preprotachykinins gene related proteins are regulated and foster better mechanistic knowledge to support the development of new medicines to treat neuropathic and chronic pain.