*4.7. LC-MS*/*MS Analysis*

The extraction of proteins from decellularized and repopulated lung tissue sca ffolds was modified after Rosmark et al. [13]. Instead of consecutive protein extraction we here performed one protein extraction procedure. The spanned tissue area of the sca ffolds (decellularized or repopulated with heavy labeled cells) were lyophilized, diluted in extraction bu ffer with 100 mM ammonium bicarbonate with 8 M urea, and homogenized using a Bioruptor ®Plus (Diagenode SA, Seraing, Belgium) at 4 ◦C for 20 cycles 15 s ON/OFF. Samples were reduced with 5 mM TCEP (tris-2-carboxyethyl phosphine) 30 min at 37 ◦C at 850 rpm, alkylated with 10 mM IAA (iodoacetamide) for 45 min at room temperature, followed by overnight trypsin digestion at 37 ◦C at 300 rpm. Decellularized sca ffolds samples were desalted with C18 reversed-phase spin columns (Harvard Apparatus, Holliston, MA, USA) according to manufacturer's instructions, whereas repopulated sca ffolds were desalted using SOLAμ™-SPE plates (Thermo Fisher Scientific) according to manufacturer's instructions.

After desalting, samples were resuspended in 2% acetonitrile, 0.1% formic acid and the peptide concentrations were measured using Pierce ™ Quantitative Colorimetric Peptide Assay (Thermo Scientific, Rockford, IL, USA). For all samples we adjusted the volume to inject 1 μg peptides. Peptide separations and data acquisitions were performed as previously described [13]. Briefly samples were separated on a 25 cm EASY-spray column using an EASY-nLC 1000 LC-system (Thermo Fischer Scientific) using a gradient of 5%–30% bu ffer B over 60 min and 30%–95% bu ffer B for 5 min with a flow rate of 300 nL/min. Data were acquired with a Q Exactive Plus mass spectrometer (Thermo Fischer Scientific) using top-15 data dependent acquisition (DDA) where each full mass scan covered 400–1600 *m*/*z* at resolution 70,000 at 200 *m*/*z* for both MS and MS/MS scans. MS precursor values above 1.7 × 10<sup>4</sup> were required for triggering MS/MS scans. An automatic gain control (AGC) of 1 × 10<sup>6</sup> with ion accumulation time of 100 ms for MS scans and 60 ms for MS/MS was used.
