TiO2 Phosphopeptide Enrichment

Phosphopeptides were enriched over TiO₂ (Titanium dioxide). A ratio of 10:1 TiO₂ beads/protein were resuspended into 100 μl methanol and centrifuged at 2,500 × g at room temperature for 2 min to discard methanol. TiO₂ beads were subsequently washed once with 100 μl of 1% v/v ammonia solution for 10 min with vortex mixing and centrifuged at 2,500 × g at room temperature for 2 min to discard solution. TiO₂ beads were then equilibrated with 50 μl of 1 M glycolic acid in 80% v/v ACN and 6% v/v TFA for 60 s and centrifuged at 2,500 × g at room temperature for 2 min to discard solution. Two hundred microliter digested peptides were mixed with equilibrated TiO₂ beads for 30 min incubation with continuous mixing and centrifuged at 2,500 × g at room temperature for 2 min to discard solution. Peptides and TiO₂ beads mixture were washed once with 100 μl of 1 M glycolic acid in 80% v/v ACN and 6% v/v TFA for 30 s and centrifuged at 2,500 × g at room temperature for 2 min to discard solution. Peptides and TiO₂ beads mixture were then washed two times with 100 μl of 80% v/v ACN and 1% v/v TFA for 2 min and centrifuged at 2,500 × g at room temperature for 2 min to discard solution. Enriched peptides were eluted from TiO₂ beads three times with 1% v/v ammonia solution and 15 min incubation. Eluates were immediately acidified with 70 μl of 10% v/v formic acid.