Offgel fractionator

Three samples were ready for fractionation as mentioned previously. Each sample contains 200 μg of peptides digest. Samples were then brought to 360 μL using HPLC water, then four volumes of OFFGEL running buffer stock solution were added to each sample. OFFGEL running buffer stock solution was prepared by mixing 60 μL of the OFFGEL buffer and 300 μL of 50% glycerol brought up to 50 mL HPLC water. Three IPG frozen strips pH: 3–10 were placed in the IPG plastic tray and plastic frame cups then were placed on top of the IPG strips. IPG strips were rehydrated using 40 μL rehydration solution added to each cup. Rehydration solution was prepared by mixing 4:1 OFFGEL running buffer stock and HPLC water. Swirling strips were then incubated for 15 min. Wet electrode pads were then placed on each edge of the IPG strips. 150 μL of each sample was then loaded into each cup for all 12 cups and cups then were sealed with a silicon lid. Mineral oil was added at the anode and cathode ends and then the electrodes were placed on the IPG strips and connected to the instrument (Agilent, Santa Clara, CA, USA, G3100A, OFFGEL fractionator) where fractionation was performed for 24 h (37 (link), 38 (link)). The fractions were then harvested and stage tipping protocol was performed.

Samples of EVs were re-constituted in buffer containing 8 M urea and 0.1 M Tris-HCl. Protein concentration of the EVs lysate was measured using a Bradford assay (Bio-Rad, Hercules, CA). Protein samples (250 μg) were reduced by treatment with 5 mM Tris(2-carboxyethyl)phosphine (Pierce, Rockford, IL, USA) at 37°C for 30 min and alkylated with 15 mM iodoacetamide at room temperature for 60 min. Then, the samples were digested with mass-spectrometry grade trypsin gold (Promega, Rockford, IL, USA) at 37°C overnight. Peptides were desalted on a Sep-Pak C18 cartridge (Waters, Milford, MA, USA) and separated into 5 fractions based on their isoelectric point via OFFGEL fractionator (Agilent Technologies, Santa Clara, CA, USA). Each peptide fraction was analyzed using a high-performance liquid chromatography (HPLC)-chip/quadrupole time-of-flight system (Agilent Technologies). The chromatography parameters were as follows: 160 nL enrichment column; 75 μm × 150 mm separation column packed with Zorbax 300SB-C18 (5 μm); flow rate, 0.4 μL/min. Eluted peptides were selected for collision-induced dissociation during alternative events of an MS scan over the m/z range of 300–2400 at the rate of 4 spectra/s, and an MS/MS scan over the range of 100–3000 m/z at 3 spectra/s.