SDS-PAGE Based Extraction of Extracellular Vesicles Associated Proteins: A Procedure to Extract Proteins from EVs and Prepare Them for In-Gel Digestion

1. Characterization of EVs

1. Western Blot Analysis
   1. Protein extraction
      1. Isolate the EVs by size exclusion chromatography column (SEC) and quantify using nanoparticle tracking analysis (NTA).
      2. Mix 50 μL of RIPA buffer (150 mM sodium chloride [NaCl], 50 mM Tris, 5 mM Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid [EGTA], 2 mM Ethylenediamine-tetraacetic acid [EDTA], 100 mM sodium fluoride [NaF], 10 mM sodium pyrophosphate, 1% Nonidet P-40, 1 mM Phenylmethanesulfonyl fluoride [PMSF], 1x protease inhibitor) with the EV sample (25 μL resulting from the EV concentration on filter) for 5 min on ice to extract proteins.
      3. Sonicate for 5 s (amplitude: 500 W; frequency: 20 kHz), 3 times on ice.
      4. Remove vesicular debris by centrifugation at 20,000 x g for 10 min, at 4 °C.
      5. Collect the supernatant and measure the protein concentration with the Bradford protein assay method.
   2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting
      1. Mix protein extracts (30 μg) with 5c Laemmli sample buffer (v/v).
      2. Load the protein mix on a 12% polyacrylamide gel.
      3. Migrate the proteins in the gel with TGS buffer (25 mM Tris pH 8.5, 192 mM Glycine, and 0.1% SDS), at 70 V for 15 min and 120 V for 45 min.
      4. Transfer the proteins onto a nitrocellulose membrane with semi-dry system at 230 V for 30 min.
      5. Saturate the membrane for 1 h at RT with blocking buffer (0.05% Tween 20 w/v, 5% milk powder w/v in 0.1 M PBS, pH 7.4).
      6. Incubate the membrane overnight at 4 °C with mouse monoclonal anti-human heat-shock protein 90 (HSP90) antibody diluted in blocking buffer (1:100).
      7. Wash the membrane three times with PBS-Tween (PBS, 0.05% Tween 20 w/v) for 15 min.
      8. Incubate the membrane for 1 h at RT with goat horseradish peroxidase-conjugated anti-mouse IgG secondary antibody diluted in blocking buffer (1:10,000).
         ​NOTE: A negative control is performed using secondary antibody alone.
      9. Repeat the washing step (step 1.1.2.7).
      10. Reveal the membrane with enhanced chemiluminescence (ECL) western blotting substrate kit (see Table of Materials).

2. Proteomic Analysis

1. Protein extraction and in-gel digestion
   1. Isolate the EVs by size exclusion chromatography column (SEC) and quantify using nanoparticle tracking analysis (NTA). Repeat step 1.1.1 for EV protein extraction.
   2. Perform protein migration in the stacking gel of a 12% polyacrylamide gel.
   3. Fix the proteins in the gel with Coomassie blue for 20 min at RT.
   4. Excise each colored gel piece and cut it into small pieces of 1 mm³.
   5. Wash the gel pieces successively with 300 μL of each solution: ultrapure water for 15 min, 100% acetonitrile (ACN) for 15 min, 100 mM ammonium bicarbonate (NH₄HCO₃) for 15 min, ACN:100 mM NH₄HCO₃ (1:1, v/v) for 15 min, and 100% ACN for 5 min with continuous stirring.
   6. Dry completely gel pieces with a vacuum concentrator.
   7. Perform protein reduction with 100 μL of 100 mM NH₄HCO₃ containing 10 mM dithiothreitol for 1 h at 56°C.
   8. Perform protein alkylation with 100 μL of 100 mM NH₄HCO₃ containing 50 mM iodoacetamide for 45 min in the dark at RT.
   9. Wash the gel pieces successively with 300 μL of each solution: 100 mM of NH₄HCO₃ for 15 min, ACN:20 mM NH₄HCO₃ (1:1, v/v) for 15 min, and 100% ACN for 5 min with a continuous stirring.
   10. Completely dry the gel pieces with a vacuum concentrator.