Protein Markers of Ischemic Insult in Brain Endothelial Cells Identified Using 2D Gel Electrophoresis and ICAT-Based Quantitative Proteomics

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• 作者：Arsalan S. Haqqani ; John Kelly ; Ewa Baumann ; Reiner F. Haseloff ; Ingolf E. Blasig ; Danica B. Stanimirovic
• 刊名：Journal of Proteome Research
• 出版年：2007
• 出版时间：January 2007
• 年：2007
• 卷：6
• 期：1
• 页码：226 - 239
• 全文大小：543K
• 年卷期：v.6,no.1(January 2007)
• ISSN：1535-3907

文摘

The blood-brain barrier (BBB) is formed by endothelial cells of cerebral microvessels sealed by tightjunctions. Ischemic brain injury is known to initiate a series of biochemical and molecular processesthat lead to the disruption of the BBB, development of vascular inflammation, and subsequentneurovascular remodeling including angiogenesis. Molecular effectors of these changes are multipleand are regulated in a dynamic fashion. The current study was designed to analyze changes in cellularand secreted proteins in rat brain endothelial cells (BEC) exposed to ischemic insult in vitro using twocomplementary quantitative proteomic approaches: two-dimensional gel electrophoresis (2DE) andisotope-coded affinity tag (ICAT)-based proteomics. We show a comprehensive qualitative andquantitative comparison between the two proteomic methods applied to the same experimental systemwith respect to their reproducibility, specificity, and the type of proteins identified. In total, >160 proteinsshowed differential expression in response to the ischemic insult, with 38 identified by 2DE and 138 byICAT. Only 15 proteins were commonly identified. ICAT showed superior reproducibility over 2DE andwas more suitable for detecting small, large, basic, hydrophobic, and secreted proteins than 2DE.However, positive identification of proteins by MS/MS was more reliably done using a 2DE-basedmethod compared to ICAT. Changes in proteins involved in nucleic acid, protein, and carbohydratemetabolism, signal transduction, cell structure, adhesion and motility, immunity and defense, cell cycle,and apoptosis were observed. The functional significance of observed protein changes was evaluatedthrough a multifaceted protein classification and validation process, which included literature miningand comparative evaluation of protein changes in analogous in vitro and in vivo ischemia models. Thecomparative analyses of protein changes between the in vitro and in vivo models demonstrated asignificant correlative relationship, emphasizing the 'translational' value of in vitro endothelial modelsin neurovascular research.