107 4 Proteomic signature of macrophages cultured on collagen type 1 determined with a microplate BCA protein assay following the manufacturer’s instructions (Pierce™ Microplate BCA Protein Assay Kit, Thermo Fisher Scientific). For tryptic in-solution digestion, 50 µg of the samples was diluted in 0.1 M TEAB (pH 8.3) to a final volume of 100 µL. For reduction, the samples were incubated with 10 mM dithiothreitol (DTT, dissolved in 0.1 M TEAB, pH 8.3) on a shaker (600 rpm) at 57 °C for 10 minutes. Afterwards, the samples were alkylated with 20 mM iodacetamide (0.1 M TEAB, pH 8.3) and incubated in the dark at room temperature for 30 minutes. Subsequently, free iodoacetamide was quenched by adding 10 mM of DTT (0.1 M TEAB, pH 8.3). For tryptic digestion, the samples were incubated in 1 µL trypsin solution (0.5 µg/µL sequencing grade modified trypsin, dissolved in trypsin resuspension buffer (Promega, Walldorf, Germany) at 37 °C for 16 hours. Afterwards, the samples were acidified by adding formic acid to a final concentration of 1% and centrifuged at 16,000 g at room temperature for 5 minutes. The supernatants were then transferred to a new reaction vial and evaporated to dryness. Liquid chromatography (LC)-mass spectrometry (MS)/MS analysis in data-dependent acquisition mode For data independent analysis (DIA), a macrophage-specific protein/peptide library of the alveolar macrophages was generated using data dependent acquisition (DDA) mode. For this purpose, 100 µg of a dried tryptic digest of an alveolar macrophage reference sample was separated by high pH reversed-phase chromatography using a Dionex Ultimate 3000 ultra-performance liquid chromatography (UPLC) system equipped with a fraction collector. Peptides were separated with a C18 column (ACQUITY UPLC BEH C18 Column, 130 Å, 1.7 µm, 1 mm × 150 mm, Waters, Manchester, UK, buffer A: 5% acetonitrile (ACN), dissolved in HPLC-H20, pH 9.5 adjusted with NH4OH; buffer B: 95% ACN, dissolved in HPLC-H20, pH 9.5 adjusted with NH4OH) using a gradient from 1-50% buffer B in 60 minutes and a flow-rate of 100 µL/min. 30 fractions were collected with a volume of 200 µL. The fractions were pooled into 13 fractions and evaporated to dryness. For LC-MS/MS analysis in data-dependent acquisition mode, the samples were dissolved in 20 µL of 0.1% formic acid and 1 µL was injected into a nano-ultra pressure liquid chromatography system (Dionex UltiMate 3000 RSLCnano pro flow, Thermo Fisher Scientific, Bremen, Germany) coupled via electrospray-ionization source to a tribrid orbitrap mass spectrometer (Orbitrap Fusion Lumos, Thermo Fisher Scientific, San Jose, CA, USA). The samples were loaded (15 µL/min) on a trapping column (nanoE MZ Sym C18, 5 μm, 180 µm × 20 mm, Waters, Eschborn, Germany, buffer A: 0.1% formic acid in HPLC-H2O; buffer B: 80% acetonitrile, 0.1%
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