Figure 2

Single-step purification of thiophosphopeptides. (a) General scheme for thiophosphospeptide isolation. Proteins were labeled with cyclin A-CDK2 (F80A) and PE-ATP-γ-S and subjected to tryptic digest. The resulting peptides were mixed with disulfide beads, which capture both thiophosphopeptides and cysteine-containing peptides. The beads were then treated with basic solution to selectively release only the phosphopeptides. (b) The chemistry underlying the thiophosphopeptide selectivity. Both thiophosphate and cysteine moieties contain reactive thiol groups that can be covalently captured by disulfide beads. At high pH values, the phosphorothiolatesulfide linkages (near the upper arrow) are hydrolyzed to allow the release of the bead-bound peptides while the alkyldisulfide linkages (near the lower arrow) are stable and thus peptides are retained on the beads. Note that during the hydrolysis of the phosphorothiolatesulfide linkage, thiophosphate is converted to normal phosphate.