Tmtpro 16plex isobaric label reagent set

Peptides were labeled with tandem-mass tag (TMT) isobaric labels (TMTpro 16plex™ Isobaric Label Reagent Set, Lot UL296296, Thermo Fisher Scientific). Ten micrograms of each sample were aliquoted into a final volume of 100 µL of 100 mM TEAB, and peptides were labeled according to the manufacturer’s protocol. Multiplexed samples were fractionated by high pH reversed-phase chromatography (1260 Infinity II, Agilent Technologies) as previously described [23 (link)]. Each multiplex contained a TMT channel of pooled samples specific for a given brain region, a channel of pooled samples representing all the brain regions and 14 channels corresponding to 14 individual patient samples. Twenty-four concatenated fractions were generated for global LC-MS/MS analysis.

Samples were labeled with tandem mass tag (TMT) reagents using the TMTpro 16plex Isobaric Label Reagent Set (Thermo Fisher Scientific, A44521). Briefly, TMT label reagents were equilibrated to RT and dissolved in 30 µL of anhydrous acetonitrile, of which 10 µL were added to 25 µL of the respective sample (pH 8.0–8.5). Labeled samples were incubated for 1 h at RT and 600 rpm. To quench the excess of reactive TMT labeling reagents, 4 µL of 1 M TRIS at pH 8.0 were added and incubated for 15 min at RT and 600 rpm. Samples were equally mixed, 5% formic acid were added, dried in the SpeedVac at RT and high vacuum for about 2.5 h and then stored at −20°C until further use.