Tmt10plex label reagent

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.

Peptides from the CSF samples were labeled using TMT 10-plex™ Label Reagent set as per the manufacturer’s instruction (Thermo Fisher Scientific, USA). Briefly, before use, the TMT labeling reagents were removed from the freezer and stabilized at room temperature (approximately 30 min). Then, 41 μL of ACN was added to each channel, dissolved through vortexing, centrifuged, and set aside. The dissolved reagent was added to 100 μg of sample (i.e., mix 1:1). The solution was allowed to stand for at room temperature for 1 h. After adding 8 μL of hydroxylamine, the solution was incubated at room temperature for 15 min to terminate the reaction. The samples of each group from 10 channels were combined separately, and the vortexed to allow complete mixing. Salt and other impurities were removed from the sample before freezing at -80°C.