School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore.
J Proteome Res. 2010 Jan;9(1):472-84. doi: 10.1021/pr900829h.
Cerebral ischemia is a major cause of death and long-term disability worldwide. Ischemic penumbra, the electrically silent but metabolically viable perifocal brain tissue, is the target for the much elusive stroke therapy. To characterize the molecular events of the dynamic penumbra, we applied an iTRAQ-based shotgun proteomic approach in an in vitro neuronal model, using the rat B104 neuroblastoma cell line. Various functional and cytometric assays were performed to establish the relevant time-point and conditions for ischemia to recapitulate the pathology of the penumbra. Two replicate iTRAQ experiments identified 1796 and 1566 proteins, respectively (<or=1.0% false discovery rate). Mining of proteomic data indicated the up-regulation of proteins involved in ammoniagenesis, antiapoptotic, anti-inflammatory and mitochondrial heat shock response and down-regulation of proteins pertaining to antioxidative defense and protein metabolism. Additionally, many proteins (for instance, park7 and VAP-A) involved in the chronic neurological disorders (such as Alzheimer's disease, Parkinson's disease or Bipolar disorder) were also regulated in this model of acute neuronal injury. Our results also provide preliminary evidence about the presence of a relative glucose paradox under in vitro conditions indicating possible application of this cell system to study the mechanisms of transient protection induced by concomitant glucose deprivation under hypoxia. In conclusion, our study shows the potential application of iTRAQ-based quantitative proteomics for the elucidation of pathophysiology and the discovery of novel therapeutic targets in the field of neuroproteomics.
脑缺血是全球范围内导致死亡和长期残疾的主要原因。缺血半暗带是电沉默但代谢活跃的周围脑组织，是难以捉摸的卒中治疗的靶点。为了描述动态半暗带的分子事件，我们应用基于 iTRAQ 的鸟枪法蛋白质组学方法，在体外神经元模型中，使用大鼠 B104 神经母细胞瘤细胞系。进行了各种功能和细胞计量测定，以建立复制半暗带病理的缺血相关时间点和条件。两个重复的 iTRAQ 实验分别鉴定到 1796 和 1566 种蛋白质（<或=1.0%假阳性率）。蛋白质组学数据分析表明，氨生成、抗凋亡、抗炎和线粒体热休克反应相关蛋白上调，抗氧化防御和蛋白质代谢相关蛋白下调。此外，许多涉及慢性神经紊乱（如阿尔茨海默病、帕金森病或双相情感障碍）的蛋白质（例如 park7 和 VAP-A）也在这个急性神经元损伤模型中被调节。我们的结果还提供了初步证据，表明在体外条件下存在相对葡萄糖悖论，这表明该细胞系统可能适用于研究缺氧时伴随葡萄糖剥夺诱导的短暂保护机制。总之，我们的研究表明，基于 iTRAQ 的定量蛋白质组学在神经保护蛋白质组学领域中的病理生理学阐明和新治疗靶点的发现方面具有潜在的应用。
