Patent ID: 7423132

Claim:
A method for the separation of a phosphorylated peptide from a complex protein mixture which comprises: (a) providing a cell extract comprising a complex protein mixture derived from cell lysis; (b) digesting the protein mixture in the cell extract to provide a peptide mixture comprising one or more of the phosphorylated peptide; (c) drying the peptide mixture; (d) protecting any carboxylate groups in the peptide mixture of step (c) by reaction with an esterification agent or an amination agent or a thiolating agent; (e) reacting the mixture of step (d) with a diazo group—substituted organic moiety linked to a separation medium to bind a phosphate group of the phosphorylated peptide to the organic moiety in place of the diazo group; (f) separating the phosphorylated peptide bound to the separation medium from the mixture; and (g) releasing the bound phosphorylated peptide from the separation medium.