Noben Jean-Paul, Dumont Debora, Kwasnikowska Natalia, Verhaert Peter, Somers Veerle, Hupperts Raymond, Stinissen Piet, Robben Johan
Biomedical Research Institute, Hasselt University, and School of Life Sciences, transnationale Universiteit Limburg, Diepenbeek, Belgium.
J Proteome Res. 2006 Jul;5(7):1647-57. doi: 10.1021/pr0504788.
Neurological diseases, including multiple sclerosis (M.S.), often provoke changes in the functioning of the endothelial and epithelial brain barriers and give rise to disease-associated alterations of the cerebrospinal fluid (CSF) proteome. In the present study, pooled and ultrafiltered CSF of M.S. and non-M.S. patients was digested with trypsin and analyzed by off-line strong cation-exchange chromatography (SCX) coupled to on-line reversed-phase LC-ESI-MS/MS. In an alternative approach, the trypsin-treated subproteomes were analyzed directly by LC-ESI-MS/MS and gas-phase fractionation in the mass spectrometer. Taken together, both proteomic approaches in combination with a three-step evaluation process including the search engines Sequest and Mascot, and the validation software Scaffold, resulted in the identification of 148 proteins. Sixty proteins were identified in CSF for the first time by mass spectrometry. For validation purposes, the concentration of cystatin A was determined in individual CSF and serum samples of M.S. and non-M.S. patients using ELISA.
包括多发性硬化症（M.S.）在内的神经系统疾病，常常引发脑内皮和上皮屏障功能的变化，并导致与疾病相关的脑脊液（CSF）蛋白质组改变。在本研究中，将多发性硬化症患者和非多发性硬化症患者的脑脊液进行合并和超滤，用胰蛋白酶消化，并通过离线强阳离子交换色谱（SCX）与在线反相液相色谱-电喷雾串联质谱（LC-ESI-MS/MS）联用进行分析。在另一种方法中，经胰蛋白酶处理的亚蛋白质组直接通过液相色谱-电喷雾串联质谱（LC-ESI-MS/MS）和质谱仪中的气相分级分离进行分析。综合来看，这两种蛋白质组学方法与包括搜索引擎Sequest和Mascot以及验证软件Scaffold在内的三步评估过程相结合，共鉴定出148种蛋白质。通过质谱首次在脑脊液中鉴定出60种蛋白质。为进行验证，使用酶联免疫吸附测定法（ELISA）测定了多发性硬化症患者和非多发性硬化症患者个体脑脊液和血清样本中胱抑素A的浓度。
