Amicon ultra 10k filter

EV proteins (30 µg) were lyophilized and solubilized in lysis buffer (50 mM Tris-HCl, 1% NP-40, 0.25% Na-deoxycholate, 100 mM NaCl, 1 mM EDTA, pH 7.5) with protease inhibitor cocktail (Roche, Mannheim, Germany) and then denatured with 6 M guanidine-HCl and 50 mM tris(2-carboxyethyl)phosphine for 5 min at 95ºC. The denatured proteins were alkylated with 50 mM iodoacetamide for 30 min at room temperature in the dark. After alkylation, proteins were precipitated by methanol/chloroform precipitation and then resuspended with 2 M urea in 50 mM NH₄HCO₃. Trypsin (enzyme to protein ratio 1:100) was treated for 16 h at 37ºC. The tryptic peptides were collected via the Amicon Ultra 10 K filter (Millipore, Temecula, CA). The residual undigested proteins in the filter were additionally digested with trypsin (enzyme to protein ratio 1:100) in 50 mM NH₄HCO₃ for 6 h at 37ºC. Additional tryptic peptides were collected and the filter was rinsed with 500 mM NaCl and water. All eluents from the filter were combined and desalted with the C18 Spin Columns (Thermo Fisher Scientific, Rockford, IL).

Aliquots of 50 µg were prepared from supernatants by filter aided sample preparation (FASP) method as follows: protein extracts were diluted in 8 M urea to a final concentration of 0.5 µg/µL, reduced with 0.1 volume of 50 mM DTT and alkylated with 0.1 volume of 100 mM iodoacetamide. The protein extract was then transferred into the Amicon Ultra 10K filter (Millipore, Darmstadt, Germany) and washed twice with 8 M urea and then twice with 25 mM ammonium bicarbonate. Trypsin was added at a 1:50 ratio for a final concentration of 10 ng/µL. Peptides were purified using a 1 mL 30 mg Hydrophilic-Lipophilic-Balanced (HLB) cartridge following the manufacturer’s protocols (Waters, Milford, MA, USA) and finally filtered using 0.22 µm spin columns (Agilent Technologies, Santa Clara, CA, USA).