Supplemental Data Phosphatidylinositol-(4,5)-Bisphosphate Regulates Sorting Signal Recognition by the Cathrin-Associated Adaptor Complex AP2

Sample preparation and tryptic digestion of the phosphorylated and nonphosphorylated AP2 complex for mass spectrometry. AP2 samples were stored at 4°C and buffer-exchanged twice with 200 mM ammonium acetate, pH 7.5 using microcentrifuge columns (Micro Bio-spin 6, Bio-Rad) before analysis. Buffer exchange and sample introduction were conducted at room temperature. Dissociation of the complex was performed by treating the sample in 200 mM ammonium acetate at pH 7.5 with a cation exchange matrix (AG 50W-X8 matrix, Bio-Rad). This treatment effectively reduces the pH to between 2 and 3 and this causes the subunits to dissociate. Tryptic digestion was performed on samples of phosphorylated and nonphosphorylated AP2 buffer exchanged to 200 mM ammonium acetate, pH 7.5 and at a complex concentration of 1.5-2.0 mg/ml, estimated by measuring the absorbance at 280 nm. Proteolysis was initiated by adding a stock solution of bovine pancreatic tryspin at a protein/enzyme ratio of 1/50 by weight. The digestions were performed at 37°C in a thermostated bath and stopped after 1 hour by dilution of aliquots of the reaction mixture 2fold with a solution of 3% acetic acid in acetonitrile.