Tmt isobaric mass tag labeling kit

Total protein was extracted using a protein extraction buffer consisting of 7 M urea, 2 M Thiourea, 4% Chaps, 1%DTT, and 0.5% (v/v) protease inhibitor cocktail. The protein concentration was determined by Bradford method using bovine serum albumin as the standard. According to the manufacturer instruction of TMT Isobaric Mass Tag Labeling kit (Thermo, Germany), two samples of miR-34b-3 and negative control (100 μg of each sample) were dissolved in 45 μl of 100 mM triethyl ammonium bicarbonate (TEAB), added with 5 μl 200 mM TCEP and incubated at 55°C for 60 min. 5 μl of 375 mM iodoacetamide (IAA) was added to samples and incubated for 30 min room temperature in the dark. Protein lysates were precipitated with acetone and digested overnight at 37°C with 2.5 μl (1 μg/μl) of Trypsin. Each 100 μg sample was labeled with 41 μl of the TMT Label Reagents. The two-plex TMTs Label Reagents were equilibrated to room temperature and each aliquot was resuspended in 41 μl of anhydrous acetonitrile. Samples were respectively labeled as follows: TMT-129, NC; TMT-130, miR-34b-3. After reaction for 60 min at RT, each tube was added with 8 μl of 5% hydroxylamine and incubated for 15 min. Finally, samples were pooled and desalinated for LC-MS/MS analysis.

Different mixed samples from the same group (4 samples of NBD-NFPA fibroblasts group and 4 samples of BD-NFPA fibroblasts group) at a ratio of 1:1:1:1, individually, and measured for concentrated low-abundance proteins with ProteoMiner protein enrichment kit (Bio-Rad, California, USA). Total protein was extracted using a protein extraction buffer consisting of 7 M urea, 2 M Thiourea, 4% Chaps, 1% DTT, and 0.5% (v/v) protease inhibitor cocktail. According to the manufacturer’s instructions of TMT Isobaric Mass Tag Labeling kit (Thermo, USA), protein pellets (100 μg of each sample) were resuspended in 100 mM triethylammonium bicarbonate (TEAB) with 200 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and incubated for 1 h at 55 °C. After addition of 5 μL of 375 mM iodoacetamide (IAA), the samples were incubated for 30 min at room temperature in the dark, then digested overnight with trypsin at 37 °C (Promega). Each 100 µg sample was labeled with 41 µl of the TMT Label Reagents following the manufacturer’s protocol. The NBD-NFPA fibroblasts sample was labeled with reporter tag 126, whilst the BD-NFPA fibroblasts sample was labeled with reporter tag 127. Following labeling, the peptide mixtures were pooled and desalinated for LC-MS/MS analysis.