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

在神经性动物中，创伤性神经组织导致痛觉过敏和异常性疼痛后的疼痛感知治疗已被广泛研究，但成功率有限。本研究计划将分三个阶段展开。初始阶段将包括在中枢神经系统中使用pre - proachykinins （pt - a）敲除和野生型小鼠进行差异蛋白质组探索，以鉴定由pre - proachykinins基因编码的特定蛋白质，重点是速激肽神经肽的前欧洲肽。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，表征和定量，特别关注在经过验证的大鼠神经性疼痛模型中的速激肽神经肽的前欧洲肽。我们在脊髓多肽和蛋白质分析方面发展了专业知识，使用特定提取/浓缩技术，消化方法，SDS-PAGE， 2D凝胶电泳，毛细管高效液相色谱，离子捕获质谱和MALDI-TOF。蛋白质定量将使用多重等压标记技术进行生物标志物发现，以表征蛋白质表达比例谱，进行主成分分析（PCA），并定义多参数谱，主要集中在用于诊断和预后目的的前体裂蛋白基因相关蛋白。此外，第三个目标将包括评估瞬时受体电位通道香草蛋白1 （TRPV1）激动剂/拮抗剂施用后的神经蛋白质组调节。TRPV1受体参与疼痛的传递和调节，TRPV1配体在缓解疼痛方面显示出良好的效果。本提案的总体目标是了解神经蛋白的机制和条件，特别是对前体收缩素基因相关蛋白的特殊兴趣，并促进更好的机制知识，以支持开发治疗神经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.