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.