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

在神经病动物中，创伤性神经组织导致痛觉过敏和异常性疼痛之后的疼痛感知的治疗已经被广泛研究，但成功有限。这项研究计划将分三个阶段进行。初始阶段将包括在中枢神经系统中使用前速激肽原（PPT-A）基因敲除小鼠和野生型小鼠进行差异蛋白质组探索，以鉴定前速激肽原基因编码的特异性蛋白质，重点是速激肽神经肽的前神经肽。第二阶段将包括中枢神经系统中的系统性蛋白质组探索，包括神经蛋白质组的鉴定、表征和定量，特别关注经验证的神经性疼痛大鼠模型中的速激肽神经肽的原神经肽。我们开发了一种专门的技术，用于分析脊髓中的肽和蛋白质，使用特定的提取/浓缩技术，消化方法，SDS-PAGE，2D凝胶电泳，毛细管HPLC，离子阱质谱和MALDI-TOF。将使用用于生物标志物发现的多重同量异序标记技术进行蛋白质定量，以表征蛋白质表达比率谱，进行主成分分析（PCA）并定义多参数谱，主要关注前速激肽原基因相关蛋白质，用于诊断和预后目的。此外，第三个目标将包括评价瞬时受体电位通道香草酸1（TRPV 1）的激动剂/拮抗剂给药后的神经蛋白质组调节。TRPV 1受体参与疼痛的传递和调节，TRPV 1配体显示出有希望的缓解疼痛的结果。该提案的总体目标是了解对前速激肽原基因相关蛋白具有特殊兴趣的神经蛋白的调节机制和条件，并促进更好的机制知识，以支持治疗神经性和慢性疼痛的新药的开发。

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.