Tmt pro 16 plex reagent

Tandem mass tag (TMT) labeling was performed as previously described (Zecha et al., 2019 (link)). Briefly, 100 µg peptides per sample were resuspended in 50 mM HEPES pH 8.5 at a concentration of 5 mg/ml. Dried Tandem Mass Tag (TMT) pro 16-plex reagent (ThermoFisher Scientific) was reconstituted at 20 µg/µl in 100% anhydrous MeCN and added to samples at a 2:1 TMT to peptide mass ratio. The reaction was incubated for 1 hr at 25 °C while shaking and quenched with 5% hydroxylamine to a final concentration of 0.2% for 15 min at 25 °C while shaking. The TMT-labeled samples were then combined, dried to completion by vacuum centrifugation, reconstituted in 1 ml 0.1% FA, and desalted with a 100 mg SepPak cartridge as described above.

300 μg of trypsin-digested and desalted peptides were used for each sample for isobaric labeling. Lyophilized peptides were dissolved in 20 μl 100 mM triethylammonium bicarbonate (TEAB) (Sigma). 20 μg of peptides in TEAB from each sample and replicate were additionally mixed (final volume 60 μl) as reference sample (REF). 500 μg of each TMTpro16plex reagent (Thermo Fisher Scientific) were dissolved in 30 μl of 100% anhydrous acetonitrile and added to the peptide/TEAB mixes. The REF sample was mixed with 1.5 mg (90 μl) of TMTpro16plex reagent 134N and subsequently split in 3 aliquots. Labels used: 126C: wildtype; 127C: MS-275 6 hour; 128C: MS-275 24 hour; 129C: WT HDAC1 CI; 130C: WT HDAC2 CI; 131N: HDAC1 KO; 131C: WT HDAC3 CI; 132N: HDAC2 KO; 132C: WT HDAC8 CI; 133N: HDAC3 KO; 133C: REF; 134N: HDAC8 KO. Samples were labeled for 60 min at RT. 0.1 μl of each sample were pooled, mixed with 10 μl 0.1% TFA and analyzed by mass spectrometry (MS) to check labeling efficiency. For quenching, 5 μl of 5% hydroxylamine (0.4% final concentration) were added and the reaction was incubated for 25 min at RT. Samples were pooled and subsequently desalted with Sep-Pak tC-18 (200 mg) cartridges (Waters). Desalted samples were dried for 30 min in a SpeedVac vacuum centrifuge and subsequently lyophilized overnight.