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Proteomic Strategy for Analysis of VEGF Mediated Endothelial Cell Signaling
Author(s) -
Parker Sarah Josepha,
Greene Andrew
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.746.7
Subject(s) - angiogenesis , vascular endothelial growth factor , signal transduction , biotinylation , microbiology and biotechnology , proteomics , vascular endothelial growth factor a , proteome , chemistry , kinase insert domain receptor , biology , cancer research , vegf receptors , biochemistry , gene
Vascular Endothelial Growth Factor (VEGF) is a potent and specific pro‐angiogenic cytokine, and its dysregulation has been implicated in many diseases characterized by either insufficient or excessive angiogenesis. The field of proteomics offers techniques which may greatly enhance our understanding of complex networks of protein‐protein interactions within important signaling networks such as the VEGF pathway. In the current study we examined whether a newly established technique, developed in our lab for the isolation and proteomic analysis of the TNFα signaling network, could be used to study the VEGF pathway. Recombinant rat VEGF 164 was biotinylated and administered to vascular endothelial cells for 10 minutes followed by a cell permeable chemical cross‐linker (DSP). Cell lysate was then subjected to affinity chromatography with streptavidin to isolate the VEGF pathway. Western blots for the VEGF type 2 receptor and the downstream signaling proteins Akt and ERK1/2 revealed a dose‐dependent relationship for VEGF concentration, DSP concentration, and DSP administration time, with optimal values of 250ng/mL, 0.75 mM, and 10 minutes, respectively. Control samples not treated with VEGF or DSP showed minimal expression. These data suggest that our technique is successful in isolating the VEGF pathway, and can be utilized for identification of proteins and post‐translational modifications via LC‐MS/MS.