Titanium Dioxide-based Enrichment of Phosphopeptides: A Method for the Selective Isolation of Phosphopeptides from Peptide Library

Precondition the titanium dioxide with 500 microliters of 100% ACN two times. Then, condition the titanium dioxide with 500 microliters of 0.2 M sodium phosphate buffer, pH 7, two times. Finally, use 300 microliters of equilibration buffer to wash the beads three times. Add 400 microliters of 50% ACN, 0.1% TFA to the low protein-binding tube. Then, add 84 microliters of lactic acid.

Transfer the resuspended phosphopeptides into the low protein-binding tube and incubate them at room temperature using an end-over-end rotator for 1 hour. After pelleting the beads, use 300 microliters of equilibration buffer to wash them two times and spin them down. With 300 microliters of rinsing buffer, rinse the beads two times. Then, transfer them to a 0.2-micrometer spin filter.

After spinning, transfer the filter unit to a clean 1.5-milliliter low protein-binding tube, and with 200 microliters of 0.9% ammonium in water, elute the contents two times. After checking the pH, vacuum concentrate the eluate to dryness overnight to evaporate the ammonia.