Tmtpro 16 plex kit

Peptides from each individual cell line in the study and a global pooled reference internal standard (GIS) were labeled using the TMTpro 16-plex kit (ThermoFisher Cat#A44520 Lot#UK297033). Labeling was performed essentially as previously described (93 (link), 94 ). Briefly, each sample (containing 100 μg of peptides) was re-suspended in 100 mM TEAB buffer (100 μL). The TMT labeling reagents were equilibrated to room temperature, and anhydrous ACN (256 μL) was added to each reagent channel. Each channel was gently vortexed for 5 min, and then 41 μL from each TMT channel was transferred to the peptide solutions and allowed to incubate for 1 h at room temperature. The reaction was quenched with 5% (vol/vol) hydroxylamine (8 μl) (Pierce). All 16 channels were then combined and dried by SpeedVac (LabConco) to approximately 150 μL and diluted with 1 mL of 0.1% (vol/vol) TFA, then acidified to a final concentration of 1% (vol/vol) FA and 0.1% (vol/vol) TFA. Peptides were desalted with a 200 mg C18 Sep-Pak column (Waters). Each Sep-Pak column was activated with 3 mL of methanol, washed with 3 mL of 50% (vol/vol) ACN, and equilibrated with 2×3 mL of 0.1% TFA. The samples were then loaded, washed with 2×3 mL 0.1% (vol/vol) TFA and 2 mL of 1% (vol/vol) FA. Elution was performed with 2 volumes of 1.5 mL 50% (vol/vol) ACN. The eluates were then dried to completeness.

Samples from each group were labeled with TMTpro 16plex kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. After labeling, samples were combined and fractionated by using high-pH reversed-phase stop-and-go extraction (Hp-RP StageTips). In brief, a C8 membrane was inserted into Gilson 200 μL pipet tips used as a frit. A portion of C18-AQ beads (5 μm) was packed properly into the StageTips followed by washing and conditioning by centrifugation at 1500 g for 2 min. Peptides (30 μg) were resuspended in 200 mM NH₄COOH (pH 10). Next, peptide samples were dissolved in 50 μL of 200 mM NH₄COOH (pH 10) and transferred into the StageTips followed by centrifugation at 15000 g for 2 min to elute the peptides in 6 fractions with increasing acetonitrile concentration. The fractionated samples were then dried using Speed vac and reconstituted in 0.1% FA. The resulting peptides were desalted and concentrated using EvoTips (EvoSep, Odense, Denmark).