Proteomic sample preparation

The cells or exosomes were lysed immediately with the compatible lysis buffer containing 10 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM CaCl₂, 2 mM MgCl₂, 600 mM guanidine HCl, 1% DDM, and protease inhibitor mixture (1 mM EDTA, 1 mM phenylmethanesulfonylfluoride, 1 μg/mL leupeptin, 1 μg/mL pepstatin, and 1 μg/mL aprotinin). Protein concentration was determined by the Pierce Micro BCA Kit (Thermo). The obtained tissue lysate was processed by using the SISPROT protocol as previously described. Briefly, the samples were first acidified to pH 2–3 and loaded onto 200 or 10 μL spin-tip device packed with one plug of C18 disk (3M Empore, USA) and 0.6 mg of 20 μm POROS SCX beads (Applied Biosystems, USA) in tandem. Proteins were reduced by TCEP, alkylated by IAA and digested by trypsin (TPCK-treated, Sigma-Aldrich). The digested peptides were then transferred from the SCX beads to C18 disk with 200 mM ammonium formate (pH 10) and eluted from C18 disk with ACN concentration of 80% in 5 mM ammonium formate (pH 10).