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

研究对象：我们根据他们的压力水平选择研究对象。我们用了三个类别来分类这些科目。第一类是超健康人群，他们没有身体疾病，没有精神疾病，没有药物，没有住院史。第二组是压力组，但没有身体疾病，没有精神疾病，没有药物，没有住院史。第三组是重度抑郁症患者，但目前没有身体疾病。我们通过筛选测试和半结构化访谈找到了知情同意的受试者。方法：1)用蓝葡糖和蛋白G柱去除血浆样品中的白蛋白和球蛋白。经短暂纯化浓缩后的血浆蛋白用直接枪法进行电泳和CBB染色，以确定目标条带和斑点。从凝胶中取出条带和斑点，胰酶消化，LCQ分析多肽并确定其m/z。采用Mascot分析法对数据进行分析。2)比较各类被试的质谱数据，选择应激特异性肽和抑郁特异性肽。3)利用所选肽的特异性抗体进行western blot分析。4)利用MALDI质谱分析蛋白芯片分析应力特异性图谱。结果与讨论1)BDNF、嗜铬粒蛋白A是应激特异性标记物的候选。NK细胞活性比这两种蛋白更敏感。2)大约有20种蛋白质是应激组特有的。3)疏水蛋白芯片分析显示15-20KDa分子量区域的应力特异性模式。

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.