Phosphopeptide Enrichment Using TiO2 Beads

An aliquot of the peptide mixture was used for proteome analysis, while the remaining sample was enriched for phosphopeptides using titanium dioxide beads (TiO_2, 5 μm Titansphere, GL Sciences, Japan). The TiO₂ beads were washed with 80% ACN and 6% TFA prior to incubation with the peptide mixture to a 10:1 peptide-to-beads ratio, with gentle rotation for 10 min. After incubation, the beads were spun down, and the supernatants were incubated with fresh TiO₂ beads for a second enrichment round. The procedure was repeated for five consecutive rounds. Beads were loaded onto in-house packed C₈ StageTips and washed with 80% ACN and 6% TFA. The phosphopeptides were eluted with 2 × 50 μl 20% NH₄OH, pH 10.5, in 60% ACN, and acidified with 20 μl of 20% TFA. Eluted peptides were concentrated and ACN evaporated and loaded onto C₁₈ StageTips (Rappsilber et al., 2007).