Chloroacetamide caa

A total of 6 on-bead samples (n = 3 of CTL JIMT and n = 3 of ACT1 JIMT) were submitted to the IUSM Center for proteome analysis where proteins were denatured in 8 M urea and 100 mM Tris-HCl, pH 8.5, and reduced with 5 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEPSigma-Aldrich, St. Louis, MO, USA, Cat No: C4706) for 30 min at room temperature. Samples were then alkylated with 10 mM chloroacetamide (CAA, Sigma Aldrich Cat No: C0267) for 30 min at room temperature in the dark, prior to dilution with 50 mM Tris-HCl, pH 8.5, to a final urea concentration of 2 M for trypsin/Lys-C-based overnight protein digestion at 37 °C (0.5 µg protease, mass spectrometry grade, Promega Corporation, Madison, WI, USA, Cat No: V5072). Digestions were acidified with trifluoroacetic acid (TFA, 0.5% v/v) and desalted on Pierce C18 spin columns (Thermo Fisher, Waltham, MA, USA, Cat No: 89870) with a wash of 0.5% TFA followed by elution in 70% acetonitrile 0.1% formic acid (FA).

The exosomal proteins were lysed in 50 μL of 1% (w/v) Sodium Deoxycholate (SDC) (Sigma, St. Louis, MO, USA). The samples were boiled at 95 °C for 5 min and sonicated thrice at 10 s each. The protein concentration of the isolated exosomes was determined using a Bradford assay kit. The proteins were denatured with dithiothreitol (DTT, Bio-rad) to a final concentration of 10 mM and incubated for 30 min at 50 °C. The samples were then alkylated by adding chloroacetamide (CAA, Sigma, St. Louis, MO, USA) to a final concentration of 40 mM, and incubated for 20 min in the dark at room temperature. A ratio of 1:100 of trypsin was added into the samples and incubated overnight at 37 °C in an incubator shaker. Trypsin digestion was stopped by adding formic acid (Sigma, St. Louis, MO, USA) to a final concentration of 1%. The lysate was then subjected to ethyl acetate precipitation. An equal volume of 100% water-saturated ethyl acetate (Sigma, St. Louis, MO, USA) was added to the lysate and vortexed thoroughly. The lysate was centrifuged at 14,000× g for 5 min to separate the SDC (interphase) and peptides (aqueous phase at middle layer). The aqueous phase with peptides was transferred into a new Eppendorf tube. The ethyl acetate precipitation step was repeated and the peptides were collected again. The final collected peptides were dried in a vacuum concentrator.