Research on Stress Marker in the Crebrospinal Fluid and Blood by Proteomics Analysis

主题：我们根据他们的压力水平选择研究对象。当我们对研究对象进行分类时，我们使用了三个类别。第一类是无躯体疾病、无精神疾病、无药物治疗、无住院史的超级健康人群。第二组为应激组，但无躯体疾病，无精神疾病，无药物治疗，无住院史。第三组是重度抑郁症患者，但目前没有身体疾病。方法：1）血浆标本经蓝色琼脂糖凝胶柱和蛋白G柱去除白蛋白和球蛋白。将简单纯化和浓缩的血浆蛋白用于直接鸟枪法，并用于电泳和CBB染色以确认目标条带和斑点。从凝胶上切下条带和斑点，用胰蛋白酶消化，通过LCQ分析肽并鉴定其m/z。2）比较各组受试者的masspectrophy数据，筛选出应激特异性肽和抑郁特异性肽; 3）用特异性抗体对筛选出的肽进行western blot分析，确认其蛋白质表达; 4）用MALDI质谱技术进行蛋白质芯片分析，确定应激特异性蛋白质表达模式。嗜铬粒蛋白A是胁迫特异性标记的候选者。3）疏水蛋白芯片分析显示，在15- 20 KDa分子量区域内存在应激特异性蛋白质。

Subject :We selected the research subjects according to their stress level. We used three categories when we classified the subjects. The first category is the super healthy group who have no physical diseases, no mental diseases, no medication, no history of hospitalization. The second group is the stress group but have no physical diseases, no mental diseases, no medication, no history of hospitalization. The third group is major depression patients but have no current physical diseases. We found these subjects with informed consent through screening tests and semi-structured interviews.Methods :1)Albumin and globulin were deleted from the plasma samples by blue sepharose and protein G column. The briefly purified and concentrated plasma proteins were used for direct shot gun methods and used for electrophoresis and dyed with CBB to confirm the target bands and spots. The bands and spots were also excised from the gel, digested by trypsin, and the peptides were analyzed by LCQ and identified its m/z. The data were analyzed by Mascot analysis.2)The masspectorophy data from each category of subjects were compared and stress specific peptides and depression specific peptides were selected.3)The proteins estimated by selected peptides were confirmed by western blot analysis using their specific antibodies.4)Proteinchip analysis by MALDI massspectrometry were used for stress specific patterns.Results and Discussion1)BDNF, Chromogranin A were the candidates for stress specific markers. However NK cell activities were more sensitive than these two proteins.2)Around 20 proteins were unique to the stress group.3)Hydrophobic proteinchip analysis showed stress specific patterns around 15-20KDa molecular weight areas.