Porcine trypsin

The B16F1 and B16F10 sublines of B16 murine melanoma cells were kept in culture as recommended by the American Type Culture Collection (Manassas, VA, USA) at 5% CO₂ and 37 °C (with a Heracell 150i incubator, Thermo Fisher Scientific, Waltham, MA, USA).
Cells were routinely cultured in 25 cm² flasks (TPP, Trasadingen, Switzerland), using Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g·L⁻¹d‐glucose, supplemented with 1 mm l‐glutamine and 10% fetal bovine serum (supplemented DMEM; cell culture components purchased from Sigma‐Aldrich, Steinheim, Germany). After detaching the cells with trypsin/EDTA solution (0.5 g·L⁻¹ porcine trypsin, 0.2 g·L⁻¹ EDTA·4Na in Hanks' balanced salt solution with phenol red; Thermo Fisher Scientific), the cells were counted (TC10™ Automated Cell Counter, Bio‐Rad, Hercules, CA, USA).
The B16F1 and B16F10 sublines had the same passage number (25) when the experiments began.

For the analysis of the proteomic profile at 20°C or 37°C, protein extracts were fractionated by SDS-PAGE (in a 10% SDS-polyacrylamide gel) till the whole proteome had penetrated in the resolving gel (about 1 cm of total migration). Gels were stained with Colloidal Blue Staining Kit (Invitrogen). Each proteome was excised and cut into small pieces prior to manual in-gel digestion with trypsin. Excised bands were separately detained with 50 mM ammonium bicarbonate (ABC) (Sigma-Aldrich) and 50% acetonitrile (ACN) (Fisher Chemical). Samples were then reduced with 10 mM dithiothreitol (Bio-Rad) in 50 mM ABC and alkylated with 55 mM iodoacetamide (GE Healthcare Life Sciences) in 50 mM ABC. Then, gel pieces were digested with porcine trypsin (Thermo Fisher Scientific), at a final concentration 12.5 ng/ml in 50 mM ABC, overnight at 37°C. Peptides were extracted using 100% ACN and 0.5% trifluoroacetic acid (Sigma-Aldrich), purified using a Zip Tip (Millipore, Sigma-Aldrich), and dried. Finally, samples were reconstituted in 12 μl of 0.1% formic acid in water (Fisher Chemical) and the peptides were quantified using the Qubit^® Protein Assay Kit and the Qubit^® 1.0 fluorometer.

Leptospiral EVs were treated with lysis buffer (2% SDS in 100 mM triethyl ammonium bicarbonate, TEAB) and disrupted by sonication (amplitude 35%, pulse of 10 s, and rest of 5 s, for a total of 5 min). The protein samples were reduced with 10 mM DTT at 37 °C with mild agitation for 30 min and alkylated with 40 mM iodoacetamide at RT for 30 min in the dark. The reaction was quenched by incubation with 10 mM DTT at RT for 15 min. Protein samples were incubated with 6 volumes of cold acetone at − 20 °C overnight. Subsequently, the samples were centrifuged at 12,000×g at 4 °C for 10 min. The pellet was reconstituted with 0.6 M urea in TEAB buffer and then sonicated for 10 min. The samples were digested with porcine trypsin (Thermo Scientific) in a 1:50 (w/w) ratio at 37 °C overnight. Finally, the peptide samples were completely dried in vacuo. The peptide concentrations were measured using Quantitative Fluorometric Peptide Assay (Thermo Scientific), following the manufacturer’s instruction.