Fractionated radiation alters the extracellular matrix produced by muscle-invasive bladder cancer cells

MS analysis was performed following previously described protocols (Humphries et al., 2022; J. Robertson et al., 2015). In short, 5 µg of protein was loaded onto a 4-15% agarose gel (Thermo Fisher Scientific for SDS-PAGE electrophoresis (3 min, 200V; Thermo Fisher Scientific). Protein bands were stained with InstantBlue Coomassie (Abcam), sectioned, and in-gel trypsin digested before being analysed by tandem liquid chromatography-mass spectrometry (LC-MS/MS) using an UltiMate 3000 Rapid Separation LC system (Thermo Fisher Scientific) coupled with an Orbitrap Elite Mass Spectrometer (Thermo Fisher Scientific).Following previously described protocols (Byron et al., 2015; J. Robertson et al., 2015), MS data was analysed using an in-house Mascot Server (v. 2.5.1; Matrix Sciences) with mass tolerances of 0.4 Da and 0.5 Da for precursor and fragment ions, respectively. Data was validated using Scaffold (v. 4.6.3; Thermo Fisher Scientific) with an identification threshold of 90% at the peptide level, with at least one unique validated peptide (0.1% estimated false discovery rate). Protein identification, normalisation, and fold-change and p-values calculations were performed with Protein Discoverer (v. 2.5.0.400; ThermoFisher Scientific).