Six plex tmt kit

Cells were plated in 15 cm dishes and treated with palbociclib for 2 or 7 days. Cells were lysed in cell extraction buffer containing 2% SDS, 1X PhosStop (Roche) and 1× cOmplete protease inhibitor cocktail (Roche). An aliquot of extract containing 100 µg protein was then digested by benzonase (Merck) and precipitated by acetone. The protein pellet was resuspended in digest buffer (0.1 M triethylammonium bicarbonate, pH 8.5, Sigma‐Aldrich, tandem mass tag [TMT] labelling using a six‐plex TMT kit [Thermo Fisher Scientific] and desalted. Peptides were then separated using high pH reverse phase chromatography (Waters BEH 4.6 mm × 150 mm C18 column; A, 10 mM ammonium formate, pH 9.0; B, 80% acetonitrile plus 10 mM ammonium formate, pH 9.0) into 16 fractions (Hiraga et al, 2017 (link)). Fractions were then dried under vacuum and resuspended in 5% formic acid for liquid chromatography tandem mass spectrometry (LC‐MS/MS) analysis.

After trypsin digestion, sample peptides were desalted by Strata X C18 SPE column (Phenomenex, Torrance, CA, US) and vacuum-dried. Resulting peptides were reconstituted using a six-plex TMT kit (ThermoFisher, Shanghai, China) according to its operation manual. Briefly, one unit of TMT reagent was thawed and reconstituted in acetonitrile. The peptides mixtures were then incubated for 2 h at room temperature and pooled, desalted and dried by vacuum centrifugation.
The sample peptides were fractionated into fractions by high pH reverse-phase HPLC using the Agilent 300Extend C18 column (Agilent, Shanghai, China). Briefly, sample peptides were fractionated with a gradient of 8%–32% acetonitrile (pH 9.0) over 60 min into 60 fractions. Then, all fractions were combined into 18 fractions and vacuum dried by centrifuging.

Sample peptides were desalted using a Strata X C18 SPE column (Phenomenex, Torrance, US) and were dried by vacuum. Resulting peptides were reconstituted using a six-plex TMT kit (ThermoFisher, Shanghai, China) according to its operation manual. One unit of TMTsixplex™ Isobaric Label Reagent was used to treat 100 μg of peptide samples. One unit of reagent was mixed with 41 μL of acetonitrile on the rotator for 5 min at room temperature. TMT reagent mixture was added to peptide samples and was incubated at 25 °C for 2 h. Then, the reaction was terminated by application of 5% hydroxylamine, and the labeled peptides were combined and vacuum dried.
The sample peptides were fractionated using alkaline reverse-phase HPLC with an Agilent 300 Extend C18 column (Agilent, Shanghai, China). Briefly, sample peptides were fractionated with a gradient of 2–60% acetonitrile in 10 mM ammonium bicarbonate during 80 min into 80 fractions (pH 10). All fractions were combined into 18 fractions and vacuum dried by centrifugation.