Peptide Labelling with Tandem Mass Tags
Desalted peptides were dissolved in 200 mM HEPES (pH 8.5) and peptide concentration was measured by microBCA (Thermo). 2 μg of TMT reagents (dissolved in anhydrous acetonitrile [Acros organics]) was added to every μg of peptides at a final acetonitrile concentration of 30% (v/v) and incubated at room temperature for 1 h. The TMT-10plex isobaric label reagent set (Thermo) was used for labelling. Sample labelling was as indicated in Table S3. The reaction was quenched with 0.3% (v/v) hydroxylamine (Thistle Scientific). Equal amounts of TMT-labelled samples were combined and subjected to C18 SPE then dried by speed-vac before being subjected to high pH reversed phase fractionation (HpRP) or mass spectrometry.