Purification of unphosphorylated and phosphorylated recombinant PECAM-1 cytoplasmic domain species for mass spectrometric analysis. The vector encoding PECAMcyto-FP was modified to change tyrosine residues at positions 636 and 701 of the PECAM-1 cytoplasmic domain to phenylalanine (PECAM594-711Y636,701F) and used to transform Escherichia coli. Protein expression was induced with IPTG, and PECAM594-711Y636,701F was purified as described in supplemental Figure 1. (A) Purified PECAM594-711Y636,701F was left unphosphorylated or subjected to in vitro phosphorylation by Src, Fyn, or Fer tyrosine kinase. Unphosphorylated and phosphorylated PECAM594-711Y636,701F were separated by SDS-PAGE and stained with Coomassie blue. Note that phosphorylated PECAM594-711Y636,701F migrated as 2 species with slightly higher apparent molecular weights than unphosphorylated PECAM594-711Y636,701F on SDS-PAGE. (B) (Left) Unphosphorylated and Fer kinase-phosphorylated PECAM594-711Y636,701F were subjected to ion exchange chromatography to enable purification of unphosphorylated PECAM594-711Y636,701F and separation of the 2 species of phosphorylated PECAM594-711Y636,701F (peak 1 and peak 2) from one another. (Right) Coomassie-stained SDS-PAGE gel showing the purity of unphosphorylated and phosphorylated peaks 1 and 2 of PECAM594-711Y636,701F. mAU indicates milliabsorbence units.
