Gx 271 aspeca system

About 10mg of freeze‐dried samples were reduced with NaBH₄. Pellet then underwent acid hydrolysis in 6 mol/L HCl at 100°C for 24 hours. Hydroxyproline and crosslinks were extracted from the hydrolysate using an automated solid phase extraction system (Gilson GX‐271 ASPECA system). After extraction and drying, hydroxyproline and crosslinks were analysed by UHPLC‐ESI‐MS/MS on a Thermo Dionex UHPLC and TSQ Endura triple quad mass spectrometer. Total protein was quantified in the samples using a commercially available kit (QuickZyme Biosciences, Leiden, The Netherlands). For detailed procedures, see supporting information.

Collagen matrices were freeze dried and reduced with NaBH₄. The resulting pellet was hydrolyzed with 6 mol l⁻¹ HCl at 100 °C for 24 h. An automated solid phase extraction system (Gilson GX-271 ASPECA system) was used to extract hydroxyproline and crosslinks from the hydrolysate. The samples were analyzed for hydroxyproline crosslinks by a UHPLC–ESI–MS/MS on a Thermo Dionex UPHPLC and TSQ Endura triple quadmass spectrometer as previously published^{61 (link)}. The lower limit of detection was 0.013 pmol 10 µl⁻¹.

Collagen and elastin crosslinks in the tissue samples were analysed as previously described [46 (link)]. For collagen crosslinks, briefly, 10 mg of freeze-dried tissue samples were reduced with NaBH4. The material then underwent acid hydrolysis in 6M HCl at 105 °C for 24 h. After drying and reconstituting the samples in water, hydroxyproline and crosslinks were extracted from the hydrolysate using an automated solid phase extraction system (Gilson GX-271 ASPECA system). After extraction and drying, hydroxyproline and crosslinks were analysed by UHPLC-ESI-MS/MS on a Thermo Dionex UHPLC and TSQ Endura triple quad mass spectrometer. For elastin crosslinks, 10 mg of freeze-dried samples were processed similarly to the collagen crosslinks sample treatment except for omitting NaBH4 reduction step. Collagen crosslink analysis could not be performed on the skin, heart or kidney tissues due to the challenges in isolating pure fibrotic tissues in sufficient amounts without staining and the low amount of crosslinks present in some tissues.