Exosomal Protein Isolation and Digestion

Isolated urine exosomal proteins were resuspended in 100mM triethylammonium bicarbonate (TEABC; pH 8) containing 6 M Urea, 5 mM EDTA, and 2% SDS. The proteins were chemically denatured with 10 mM DTT for 20 min at 60°C, and alkylated with 50 mM iodoacetamide for 20 min at 25°C [15 (link)]. The denatured proteins were mixed with 30% acrylamide/bisacrylamide solution, 10% ammonium persulfate and tetramethylethylenediamine. The resulting gel was cut, then the gel was washed three times with 25 mM TEABC containing 50% acetonitrile (ACN). Trypsin digestion was performed in 25 mM TEABC overnight at 37°C. The peptides were extracted from the gel through exchange with two extraction buffers consisting of 0.1% formic acid (FA) in 25 mM TEABC or 0.1% FA in 50% ACN [16 (link)]. The buffer was dried in a SpeedVac and desalted by HLB cartridge (Waters, Milford, MA, USA).