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

Phosphopeptides are short amino acid strings containing phosphorylated serine, threonine, or tyrosine residues. To isolate phosphopeptides from a peptide mix, begin by taking titanium dioxide beads in a tube. Equilibrate to condition the beads using a suitable acidified binding buffer containing a quenching agent. Add the desired concentration of peptide mixture to the bead slurry.

Incubate the mixture with continuous shaking to prevent the beads from settling down. The acidic condition promotes the phosphate group of phosphopeptides to form coordinate bonds with titanium beads. The quenching agent in the buffer reduces any unspecific binding of non-phosphorylated peptides to the beads, leaving them in the suspension.

Centrifuge to pelletize the phosphopeptide-bound beads, while the non-phosphorylated peptides remain in the supernatant. Discard the supernatant. Transfer the phosphopeptide-bound beads to a pre-assembled spin filter column. Centrifuge to remove the binding buffer and trap the phosphopeptide-conjugated beads.

Transfer the filter into a clean tube and add an ammonia-containing solution. Ammonia turns the suspension basic, causing the phosphopeptides to disengage from the beads. Centrifuge the assembly to elute and collect the unbound phosphopeptides. The titanium dioxide beads remain in the filter cartridge. Dry the phosphopeptides to remove any traces of ammonia.

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.