Peptide Labeling and Fractionation Using TMT Reagents and High pH Reversed-Phase Chromatography

The TMT Reagent 6-plex Kit (Thermo Scientific) is employed for peptide labeling. Following the manufacturer's guidelines, 100μg of peptide mixture from each sample is labeled with TMT reagents, and subsequently fractionated using the High pH Reversed-Phase Peptide Fractionation Kit (Thermo Scientific). In essence, the dried peptide mixture is first reconstituted and acidified with a 0.1% TFA solution, and then loaded onto an equilibrated high pH reversed-phase fractionation spin column. After low-speed desalting, the bound peptides are eluted with volatile high pH wash solutions of increasing acetonitrile concentrations, and then applied to a chromatography column for the collection of 10 distinct fractions of bound peptides. The collected fractions are desalted using a C18 filter and subsequently vacuum-concentrated.