Q exactive orbitrap hybrid mass spectrometer

For LFQ analysis, peptides were re-suspended in 0.1% formic acid in water and analyzed using the Q Exactive orbitrap hybrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled with the EASY-nLC 1000 system (Thermo Fisher Scientific, Bremen, Germany). Solvents A and B were 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. A 200-min gradient (from 5% to 20% solvent B over 150 min, from 20% to 35% solvent B over 30 min, 80% solvent B for 10 min, and 5% solvent B for 10 min) was used. The peptides were loaded onto a trap column (75 μm × 2 cm, 3 μm, C18, 100 Å) and ionized via an EASY-spray column (50 cm × 75 μm ID) packed with 2-μm C18 particles at an electric potential of 2.0 kV. Full MS scans were acquired in a scan range of 350–2000 Th at a resolution of 70,000 at m/z 200. The ten most abundant ions were fragmented via data-dependent MS/MS experiments with an isolation window of 2.0 Th and exclusion duration of 30 s and at a normalized collision energy of 27 for higher energy collisional dissociation (HCD). The charge state of 1 was discarded. Maximum ion injection times were 100 ms and 50 ms for full MS and MS/MS scans, respectively. The automated gain control (AGC) target value was set to 1.0 × 10⁶ for both MS and MS/MS scans.

sEV protein samples were suspended in lysis buffer supplemented with a protease inhibitor cocktail and sonicated for 20 min. BCA protein assay kit (Pierce) was used to quantify protein concentration. Samples were prepared using Filter-Aided Sample Preparation (FASP) digestion method. The digested peptides were desalted using C18 spin columns (Harvard Apparatus), and the peptides were eluted with 80% acetonitrile in 0.1% formic acid in water. Peptides were analyzed with Q-Exactive Orbitrap hybrid mass spectrometer (Thermo) and Ultimate 3000 (U)HPLC systems (Thermo) to obtain proteomics data. Samples were prepared in duplicates and total of 4 runs were performed. Proteome Discoverer software (ver. 2.5) was used to identify and quantify proteins present in sEVs and Uniprot was used to download the homo sapiens database and predict the subcellular localization of the identified proteins. Fold change was calculated based on protein abundance. Gene Ontology terms associated with the genes encoding the identified proteins were analyzed to identify and group molecular functions of differentially regulated sEV proteins (fold change > 2, p-value < 0.05).

H460 cells were infected with Ad-LacZ and Ad-LETM1 for 24 h, followed by immunoprecipitation with anti-LETM1 antibody. Samples were further analyzed by SDS-PAGE. The resultant gel lanes containing LETM1 were reduced, alkylated and digested with trypsin. The digested peptides were analyzed using Q-Exactive orbitrap hybrid mass spectrometer coupled with an EASY-nLC 1000 system (Thermo Fisher Scientific). All tandem mass spectrometry (MS/MS) spectra were acquired in a data-dependent mode for 8 most abundant peaks from the full mass spectrometry (MS) scan (m/z; 300–2000) with 27% normalized collision energy. Dynamic exclusion duration was 15 s and isolation window was set to 2.0 m/z. The MS/MS scans were acquired at a resolution of 17,500 (at m/z 200) with an automated gain control target value of 5.0 × 10⁴ and a maximum ion injection of 60 ms to get high quality of MS/MS spectrum. The acquired MS/MS spectra were searched against Uniprot Human database with the Sequest algorithm in Proteome Discoverer 1.3 (Thermo Fisher Scientific). Peptide spectra matched (PSM) number from identified proteins in each data set (Ad-LacZ and Ad-LETM1) were compared and significantly upregulated proteins were selected as a major putative interaction partners.