Proteomics in neurotoxicology

我们正在应用蛋白质组学方法解决神经病理疾病中尚未解决的问题。识别神经精神疾病的蛋白质生物标志物的方法正在设计和测试中，例如儿科患者链球菌感染后的强迫症。免疫亲和策略的组合用于从患者血清中分离蛋白质。正在比较蛋白质的质谱模式，以确定是否存在患者状态和疾病特征的统计显著特征。我们已经测试了几种分离蛋白质组的策略，这些蛋白质组大部分不含最常见的循环蛋白（白蛋白、IgG等），并使用化学计量学分析来确定影响完整蛋白质组的可重复性的变量，使用MALDI/TOF测量。第二种策略是从基因组研究中分离出与行为疾病相关的蛋白质。一种基于异常结合素的免疫亲和性策略正在被设计出来，并在现有的基因异常结合素修饰的小鼠株上进行了测试。

We are applying proteomic methodology to unresolved problems in neuropathologic diseases. Methods to identify protein biomarkers of neuropsychiatric disorders, such as the obsessive compulsive syndrome that follows streptococcal infections in pediatric patients are being devised and tested. A combination of immunoaffinity strategies is used to separate proteins from patient sera. Mass spectral patterns of proteins are being compared to determine whether there are statistically significant characteristics of patient state and the disease trait. We have tested several strategies to isolate proteome fractions that are largely free from the most common circulating proteins (albumin, IgG, etc) and are using chemometric analyses to identify variables affecting the reproducibility of the proteome fraction profile using MALDI/TOF measurements of the intact proteins. A second strategy is to isolate proteins implicated from genomic studies as being associated with behavioral diseases. An immunoaffinity strategy based on dysbindin is being devised and tested with available genetically dysbindin-gene modified mouse strains. In collaborative studies with NHGRI, the mitochondrial electron transport complex-I proteomics are being characterized. The hypothesis that deficiency in this complex results in Parkinson?s Disease is being tested. The isolation of patient derived mitochondria is being performed in NHGRI; identification of the gel isolated complex proteins is performed in LNT. In a third series of studies, we have been characterizing post-translational modifications of indoleamine-2,3-dioxygenase (IDO), an enzyme highly activated during CNS inflammation. IDO We investigated whether IDO is affected by peroxynitrite (NO-derived reactive species) using human recombinant IDO (rIDO). The detection of nitrotyrosine by immonoblotting using anti-nitrotyrosine antibody and the measurement of IDO activity in rIDO exposed to various concentrations of peroxynitrite showed that tyrosine (Tyr) residues in rIDO were nitrated and IDO activity was inhibited in a peroxynitrite dose-dependent manner. We are isolating both enzymes over-expressed in E. coli and comparing these primary structures with the same proteins affinity isolated from activated human cells in order to understand the protein regulation and post-translational modifications.