Proteomic Analysis by Liquid Chromatography-Tandem Mass Spectrometry

The lyophilized samples digested using ProteaseMAX stock (Promega) and 2 μg of trypsin. After digestion with trypsin was used μ-C18 ZipTip (Merck Millipore) for cleaning up peptide samples. After, the samples were dried in speedvac and used for the analysis.

An Easy-nLC 1200 system (Thermo Fisher Scientific Corp., Waltham, MA, United States) was coupled to an Orbitrap Fusion Lumos instrument equipped with a nanospray source (Thermo Fisher Scientific Corp., Waltham, MA, United States). Nano-LC solvents were water with 0.1% formic acid (A) and acetonitrile: water (80:20) with 0.1% formic acid (B) and the flow rate was 300 nL/min. Samples (3 μL) were injected onto a trapping column (Acclaim PepMap 0.075 mm, 2 cm, C18, 3 μm, 100 A; Thermo) in line with a Nano-LC column (Acclaim PepMap RSLC (0.075 mm, 15 cm, C18, 2 μm, 100 A; Thermo). The sample was loaded in the trap column and washed with 20 μL of solvent A at constant pressure (500 bar). After that, the sample was eluted to the column using a flow of 300 nL/min.

MS/MS analyses were conducted in the ESI⁺ mode. The instrument settings included the spray voltage at 1950 kV, capillary temperature at 300°C, and S-Lens RF level at 30%. A full-scan event was performed in profile mode over the mass range of m/z 400–1600 at a resolution of 120,000 followed by MS/MS analyses in a cycle time of 3 s. High-collision dissociation (HCD) with a normalized collision energy set at 30% was used for fragmentation. The resulting MS/MS fragment ions were detected in the mass range of m/z 100–2000 using the Orbitrap mass analyzer at a resolution of 30,000 using centroid mode. An AGC target of 5e⁴ and a maximum injection time of 54 ms were used.

A. fumigatus databank available at UniProt¹ were loaded into MaxQuant (Tyanova et al., 2016) and used to identify the protein content on EVs. A combined list of proteins identified in all independent replicates (n = 3) were generated.