Fig. 2.

VEGF fragment characterization and mass spectrometry. A and B: carrier-free VEGF₁₆₅ (100 μg/ml) was incubated with and without 20 μg/ml NE in 44 mM sodium bicarbonate buffer, pH 7.4 for 30 min at 37°C. The reaction was stopped by adding 1 μM DFP, and the samples were reduced and cysteine blocked by treatment with dithiotheritol and iodoacetamide. Samples were resolved on 12% SDS-PAGE under reducing conditions, and protein bands were visualized either by silver staining (A) or Coomassie blue staining for mass spectrometry analysis (B). VEGF bands in Coomassie blue-stained gels were excised and subjected to in-gel trypsin digestion, and then analyzed by mass spectrometry. Sequences recovered from mock-treated VEGF and NE-treated VEGF are underlined in bold.