Tmtsixplex label reagent set

TMT labeling was performed for mass spectroscopy (MS) using a TMTsixplex™ Label Reagent Set according to the manufacturer’s protocol for the 6-plex TMT kit (Thermo Fisher Scientific, USA). Briefly, 50 µL 100 mM tetraethylammonium bromide buffer was added to the myeloid histone samples and vortexed thoroughly; the labeling reaction was performed in a 1.5 mL Eppendorf tube. Subsequently, 88 µL anhydrous acetonitrile was added to the sample, vortexed for 5 min, and centrifuged. After centrifugation, 41 µL TMT reagent was added to 100 μg of the digested NP samples. The peptides were labeled with TMT-126, TMT-127, and TMT-128, respectively, for the normal NP groups, and TMT-129, TMT-130, TMT-131, respectively, for the degenerated NP groups. Finally, the mixture was oscillated, centrifuged, incubated for 1 h at RT, and incubated in 5% hydroxylamine (8 μL) for 15 min to terminate the reaction. The samples were stored after lyophilization.

TMT six-plex Label Reagent Set (Thermo, United States) was used to label the samples. The labeling reagent was placed at room temperature for enough time. Acrylonitrile ACN (Fisher Scientific, United States) was added to vial of each labeling reagent and placed at room temperature for 5 min, during which the shaking was even. The liquids in each vial were added to the protein solution containing the same mass protein, and then blended. The liquids were placed at room temperature for 2 h. Then hydroxylamine (Sigma, United States) was added to each Ep tube to terminate the reaction. Samples of equal volume are drawn from each Ep tube to the new Ep tube.