Serum Protein Profiling by TMTpro 16plex

After 56 °C sterilization for 30 min (29 (link)), 5 μl of individual or pooled serum sample was denatured in 50 μl buffer containing 8 M urea in 50 mM Tris-Cl (pH 7.4) at 32 °C for 30 min. Then the sample was reduced with 10 mM dithiothreitol at 37 °C for 1 h, followed by alkylation for 30 min with 40 mM iodoacetamide in darkness at room temperature. After 4 times dilutions with 50 mM NH₄HCO₃, trypsin (Promega, 1/50 of protein amount) was added to the sample twice for digestion at 37 °C for 12 h and additional 4 h, respectively. The peptides were acidified with 0.1% trifluoroacetic acid, desalted with a homemade C18 (Agela Technologies, Tianjin, China) column, and dried by speed-vac. For each sample, the peptides were resolved with 200 μl of 200 mM triethylammonium bicarbonate (TEAB), and 25 μg of peptide was labeled with TMTpro 16plex label reagents (Thermo Fisher Scientific, San Jose, USA) according to the recommendations of the manufacturer. The sequentially collected samples from the same patient were arranged into the same TMT, and samples from different groups were included in each TMT (as shown in Table S2, with the pooled sample as the internal standard). The samples from each TMT were combined and dried by speed-vac.