4.4. ADSC ECM Mass Spectrometry Analysis

Mass spectrometry analysis was accomplished to identify the chemical composition of the ADSC ECM. The ECM solution was prepared by sonicating in 0.1% acetic acid solution to measure the protein concentration by Bradford assay. Then, solutions containing 10 µg of ECM protein were reduced to a 10-µL mixture of 2 mM dithioerythritol, 8 M urea, and 25 mM ammonium bicarbonate, heating at 37 °C for 1 h. The free cysteine residues were prevented from disulfide bond reformation by labeling with 10 µL of 20 mM iodoacetamide for 1 h at room temperature in the dark. Prior to adding trypsin (50:1, w/w), samples were diluted to final concentrations of 1 M urea, followed by quenching the reaction with 10 µL of 1% formic acid to deactivate any unreacted reagents. Peptides of the ECM solution were purified with a mixture of 50% acetonitrile and 0.1% formic acid passed through a C18 Zip-Tip. Finally, samples were eluted with a mixture of 50% acetonitrile and 0.1% formic acid and dried in a speed vacuum device. Spectra of the samples were recorded using electrospray ionization (ESI) quadrupole time-of-flight (QUAD-TOF) MS/MS analysis (Waters SYNAPT G2, Milford, MA, USA). To validate the protein identifications and MS/MS-based peptide, protein samples were analyzed by using Mascot (Matrix Science, London, UK).