Proteasemax

The digestion was performed as outlined on the website: http://www.biotech.wisc.edu/ServicesResearch/MassSpec/ingel.htm. In short, Coomassie Blue R-250 stained gel pieces were de-stained twice for 5 min in MeOH/H₂O/NH₄HCO₃(50%:50%:100 mM), dehydrated for 5 min in ACN/H₂O/NH₄HCO₃(50%:50%:25 mM), then once more for 1 min in 100% ACN, dried in a Speed-Vac for 2 min, reduced in 25 mM DTT (dithiotreitol in 25 mM NH₄HCO₃) for 30 min at 52°C, alkylated with 55 mM IAA (iodoacetamide in 25 mM NH₄HCO₃) in darkness at room temperature for 30 min, washed twice in H₂O for 30 s, equilibrated in 25 mM NH₄HCO₃ for 1 min, dehydrated for 5 min in ACN/H₂O/NH₄HCO₃(50%:50%:25 mM), and then once more for 30 s in 100% ACN, dried again and rehydrated with 20 μL of trypsin solution (10 ng/μL trypsin Gold [Promega] in 25 mM NH₄HCO₃/0.01% ProteaseMAX w/v [Promega]). Additionally, 30 μL of digestion solution (25 mM NH₄HCO₃/0.01% ProteaseMAX w/v [Promega]) was added to facilitate complete rehydration and excess overlay needed for peptide extraction. The digestion was conducted for 3 hr at 42°C. Peptides generated from digestion were transferred to a new tube and acidified with 2.5% trifluoroacetic acid (TFA) to 0.3% final. Degraded ProteaseMAX was removed via centrifugation (max speed, 10 min) and the peptides solid phase extracted (ZipTip C18 pipette tips Millipore, Billerica, MA).

Cellular extracts were subjected to methanol and chloroform precipitation, and the precipitated protein pellets were solubilized first in 100 μl of 8 M urea for 30 min and then in 100 μl of 0.2% ProteaseMAX (Promega) for an additional 2 hours. The protein extracts were reduced and alkylated, followed by the addition of 300 μl of 50 mM ammonium bicarbonate, 5 μl of 1% ProteaseMAX, and 20 μg of sequence-grade trypsin (Promega). Samples were digested overnight in a 37°C thermomixer (Eppendorf). We used 3 μg of peptides in each Orbitrap Fusion MS analysis.

SIEVE-captured or affinity-purified EVs bound to magnetic beads were washed with 500 µL detergent-free buffer (25 mm HEPES pH 7.2, 150 mm NaCl, ±2 mm CaCl₂), transferred to a fresh Protein LoBind tube in 100 µL detergent-free buffer, and lysed on beads with a mixture of 15 µL 0.2% ProteaseMAX (Promega V2017) and 20 µL 8 m urea in 50 mm ABC for 30 min at RT. The lysate was removed from the beads and volume adjusted to 100 µL with 50 mm ABC before reduction with 5 mm DTT for 30 min at RT followed by alkylation with 15 mm IAA for 20 min at RT in the dark. Before incubation with 0.25 µg trypsin (Promega V5113) at RT overnight, samples were supplemented with additional 5 mm DTT and 1 µL of 1% ProteaseMAX solution.
trypsin digest was stopped by the addition of 0.5% TFA, and samples were further supplemented with 2% acetonitrile. Before desalting samples via C18 SPE stage tipping (33 (link)), ProteaseMAX degradation products were removed by centrifugation at 15,000g for 10 min at RT.

The propionylated protein samples were resuspended in 50 mM NH₄HCO₃ aq. with 0.1% ProteaseMAX (Promega, V2072) and digested by the digestive enzymes indicated in Supplementary Table 1–2, 10 ng/μL Trypsin Gold (Promega, V5280) with or without 10 ng/μL Glu-C (Promega, V1651) or 2 ng/μL Asp-N in 50 mM NH₄HCO₃ aq. with 0.02% ProteaseMAX at 37 °C for 3 h or overnight. Then 5% aqueous formic acid (vol/vol) was added, and the solvents were removed by Speed-Vac evaporator to obtain dried, digested samples, which were dissolved in 0.1% aqueous formic acid (vol/vol). After centrifugation (21,130 g, 10 min), the supernatant was used for LC–MS/MS analysis.