Tmt 10 plex isobaric label kit

Dithiothreitol (5 mM, catalog no. D9163, Sigma-Aldrich, USA) was added to the protein solution, and reduction was allowed to proceed at 56°C for 30 min. The protein solution was then incubated with 11 mM iodoacetamide (catalog no. V900335, Sigma-Aldrich, USA) at room temperature in darkness for 15 min. The urea concentration of the solution was then diluted to <2 M using 100 mM tetraethylammonium bromide (catalog no. 17902, Sigma-Aldrich, USA). Finally, trypsin (catalog no. V5111, Promega, USA) was added at a trypsin-to-protein mass ratio of 1:50 for the first round of digestion for overnight and 1:100 for the second round of digestion for 4 h. Peptides were desalted on a Strata X C18 SPE column (Phenomenex, USA) and vacuum dried after trypsin digestion. They were then labeled using a TMT 10-plex isobaric label kit (catalog no. 90111, Thermo, USA), according to manufacturer instructions.

Peptides were mass tagged using Thermo-Fisher’s TMT 10-plex isobaric label kit (Cat # 90061) following the included protocol. Labeled samples were pooled for analysis.