Secretome analysis of 16HBE and 16HBErs35699176 in basal conditions

16HBE and 16HBErs35699176 cells (1x106cells per well) were seeded in 6-well plates for 24 h at 37°C and 5% CO2. Cells were then washed with 3 ml serum-free medium and incubated for 48 h. At the time of the experiment, medium was replaced with fresh 3 ml non-supplemented MEM and incubated for 6 h. Cell culture supernatants were then collected and centrifuged at 12,000 rpm for 30 min to remove cell debris. Experiments consisted of 3 biological replicates. Cell culture supernatants were prepared for mass spectrometry using a modified filter-aided sample preparation (FASP) method with modifications. Digested samples were analysed by LC-MS/MS using an UltiMate® 3000 Rapid Separation LC (RSLC, Dionex Corporation, Sunnyvale, CA) coupled to an Q Exactive™ HF Hybrid Quadrupole-Orbitrap™ mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA). Peptides were selected for fragmentation automatically by data dependent analysis.The acquired MS data was analysed using Progenesis LC-MS (v4.1, Nonlinear Dynamics, Newcastle upon Tyne, UK). Features with charges ≥ +5 were masked and excluded from further analyses, as were features with less than 3 isotope peaks. The resulting peak lists were searched against the Uniprot Human database (version 20151111) using Mascot v2.5.1, (Matrix Science, Boston, USA). The Mascot results were imported into Progenesis LC-MS for annotation of peptide peaks.