Ms safe protease inhibitor cocktail

Prior to donning the tactical gear, participants were instructed to wash their forearms with running tap water for 5–10 seconds per arm, without soap. The air-dried forearms were wiped with 70% isopropyl alcohol swabs until no visible residue was observed, and air-dried (BD, Franklin Lakes, NJ, USA). Participant’s forearms were affixed with eight adhesive free Macroduct^® Sweat Collection devices, able to retain approximately 80μL each, held in place with Velcro bands, four per arm. Compression sleeves were placed over the collection devices to keep the devices in place and induce sweat production. Refer to S3 Fig for representative images of collection device setup. Following collector placement, subjects donned tactical gear as outlined in the previous section.
After completion of the treadmill protocol, excreted sweat was collected from each of the eight collectors, via transfer pipette, and pooled in a single 5mL lo-bind Eppendorf tube on ice (Hamburg, Germany). The samples were immediately aliquotted, frozen on liquid nitrogen and lyophilized overnight (S3 Fig). Proteomics aliquots (n = 7) were supplemented with MS Safe protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). All proteomics and metabolomics (n = 10) samples were stored at -80°C until analysis.

EV were lysed in a buffer containing 1% sodium deoxycholate (SDC), 100 mM TRIS, pH 8.5 with MS-SAFE protease inhibitor cocktail (Sigma-Aldrich) by ultrasonication with a QSonica probe sonicator at 4 °C. Protein concentration was estimated by microBCA (ThermoScientific). Aliquots containing 50 µg of total proteinl were diluted to 1 mg/ml with the lysis buffer and tris(2-carboxyethyl)phosphine (TCEP) and 2-chloroacetamide (CAA) were added to reach the concentrations of 10 and 20 mM respectively. Cys reduction and alkylation was achieved by 10 min heating of the sample at 80 °C. Proteins were precipitated by addition of a four-fold volume of acetone and incubation at -20 °C overnight and spinned down in the centrifuge. The protein pellet was washed twice with acetone. The pellet was then resuspended in 50 µl of the lysis buffer in a sonication bath. Trypsin (Promega, USA) was added at the ratio 1/100 w/w to the protein content and the mixture was incubated for 2 hours at 37 °C followed by the second Trypsin treatment of 1/100 w/w and left overnight at 37 °C. Proteolysis was stopped by adding trifluoroacetic acid (TFA) to 1% v/v. Precipitated SDC was removed by centrifugation. The samples were loaded onto the LC-MS instrument directly without solid phase extraction (SPE).