TiO2 Phosphopeptide Enrichment Protocol

Phosphopeptide enrichment was performed using a TiO₂ protocol adapted for label free quantitative proteomics. In short, eluates from Oasis cartridges were normalized to 1 mL with glycolic acid solution and incubated for the indicated times (see Results) at room temperature with varying volumes of TiO₂ solution (50% slurry, GL Sciences Inc., Japan). TiO₂ beads were then packed by centrifugation in equilibrated C-18 spin columns (PepClean C-18 Spin Columns, Thermo Scientific, Rockford, IL). Beads were sequentially washed with 300 μL of glycolic acid solution, 50% ACN and ammonium acetate solution (20 mM ammonium acetate pH 6.8 in 50% ACN). An extra 50% ACN wash can be also added after the ammonium acetate solution. For phosphopeptide elution, beads were incubated three times with 50 μL 5% NH₄OH for 1 min at room temperature and centrifuged. The three eluates of each fraction were pooled and acidified by addition of FA to a final concentration of 10%. Samples were then dried using a SpeedVac and pellets were stored at −80 °C.