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

The blood−brain barrier (BBB) is formed by endothelial cells of cerebral microvessels sealed by tight
junctions. Ischemic brain injury is known to initiate a series of biochemical and molecular processes
that lead to the disruption of the BBB, development of vascular inflammation, and subsequent
neurovascular remodeling including angiogenesis. Molecular effectors of these changes are multiple
and are regulated in a dynamic fashion. The current study was designed to analyze changes in cellular
and secreted proteins in rat brain endothelial cells (BEC) exposed to ischemic insult in vitro using two
complementary quantitative proteomic approaches:  two-dimensional gel electrophoresis (2DE) and
isotope-coded affinity tag (ICAT)-based proteomics. We show a comprehensive qualitative and
quantitative comparison between the two proteomic methods applied to the same experimental system
with respect to their reproducibility, specificity, and the type of proteins identified. In total, >160 proteins
showed differential expression in response to the ischemic insult, with 38 identified by 2DE and 138 by
ICAT. Only 15 proteins were commonly identified. ICAT showed superior reproducibility over 2DE and
was 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-based
method compared to ICAT. Changes in proteins involved in nucleic acid, protein, and carbohydrate
metabolism, signal transduction, cell structure, adhesion and motility, immunity and defense, cell cycle,
and apoptosis were observed. The functional significance of observed protein changes was evaluated
through a multifaceted protein classification and validation process, which included literature mining
and comparative evaluation of protein changes in analogous in vitro and in vivo ischemia models. The
comparative analyses of protein changes between the in vitro and in vivo models demonstrated a
significant correlative relationship, emphasizing the ‘translational' value of in vitro endothelial models
in neurovascular research.
Keywords: ICAT • 2D gel electrophoresis • isotope labeling • quantitative proteomics • mass spectrometry • brain
• ischemia • endothelial cells • blood−brain barrier