Protein Identification via TMT Labeling

Protein pellets were resuspended in 50 mM ammonium bicarbonate and reduced with 20 mM DTT and alkylated with 55 mM iodoacetamide. Then digestion was performed by trypsin (1:50) at 37°C overnight. HLB cartridge (Oasis, Waters, USA) was used for desalting. Anti-TMT enrichment was conducted by incubation with anti-TMT resin for 2 h at RT with end-over-end shaking. Then the resin was washed by 2 M urea in TBS (2 column volumes) followed by 0.1% SDC in TBS and TBS only (3 column volumes each). Labeled peptides were eluted from the resin with 4 column volumes of TMT elution buffer. Samples after anti-TMT enrichment were desalted by C18 reverse-phase tips (Reprosil-Pur Basic C18, 5 mm, Maisch GmbH) as previously described (41 (link), 42 (link)).