Patent ID: 7208573
Filing Date: 2007-04-24
Classification: C07K

Abstract:
1. A method of separating phosphorylated peptides from a mixture comprising phosphorylated peptides and unphosphorylated peptides, comprising the steps of: a) reacting a collection of peptides with a non-magnetic first resin, wherein some of the peptides have one or more phosphate groups, wherein the first resin is a photocleavable resin represented by the following structural formula:  wherein, n is an integer, X is —NH—, R is —H, or an amino acid, a peptide, an isotope labeled amino acid, or an isotope labeled peptide with a primary or secondary amine, and shaded circle is a bead, a pallet, a disk, capillary, a hollow fiber, a needle, a membrane, a solid fiber, a cellulose bead, a polystyrene bead, a grafted co-polymer bead, a poly-acrylamide bead, a latex bead, a dimethylacrylamide bead, or combinations thereof; wherein the primary or secondary amine groups represented by X and/or R react with the carboxylic acid groups of the peptides to form an amide bond and the phosphate groups of the peptides to form a phosphoramidate bond; thereby forming a first collection of peptides comprising unphosphorylated peptides with first resin bound carboxylic groups and phosphorylated peptides with first resin bound carboxylic acid and phosphate groups; b) selectively cleaving the first resin that reacted with the phosphate groups of the phosphorylated peptides to regenerate the phosphate groups, thereby forming a second collection of peptides comprising unphosphorylated peptides with first resin bound carboxylic groups and phosphorylated peptides with first resin bound carboxylic acid groups; c) reacting the phosphate groups of the second collection of peptides with a second resin to form a bond between the phosphorylated peptides and the second resin, wherein the second resin comprises an amino acid residue with a primary or secondary amine group, and wherein the second resin is magnetic, thereby forming a third collection of peptides comprising unphosphorylated peptides with first resin bound carboxylic groups and phosphorylated peptides with first resin bound carboxylic acid groups and second resin bound phosphate groups; d) selectively cleaving the first resin by exposing the third collection of peptides to light, thereby forming a fourth collection of peptides comprising unphosphorylated peptides that are not bound to a resin and phosphorylated peptides that are bound to the second resin; e) separating phosphorylated peptides from unphosphorylated peptides by exposing the fourth collection of peptides to a magnetic field and separating the peptides that are not bound to a resin from peptides that are bound to the second resin, thereby forming a fifth collection of peptide comprising phosphorylated peptides that are bound to the second resin.