Extracellular Matrix Proteome Profiling

Extracellular matrix proteins deposited by patient-derived fibroblasts after 4 weeks of culture were enriched by a sequential extraction of cellular and extracellular proteins as described previously [14 (link),15 (link)]. The extracted soluble and insoluble ECM samples were further processed for proteomic analysis using Thermo Fisher Scientific’s (Waltham, MA, USA) TMT10plex label reagent. The labeled samples were pooled together and fractionated into three fractions. LC-MS of the fractions were acquired using a Thermo Fisher Scientific QE HF mass spectrometer. Peptide identification and quantification were performed using Maxquant [16 (link)] searched against the human Swiss-Prot reference database (https://www.uniprot.org/). Protein levels among fibroblast groups were compared by ANOVA test followed by Tukey’s HSD test.
Additional details for Materials and Methods are provided in Supplementary Materials.