Chemically targeted positional identification of post-translationally phosphorylated peptides
AssigneeCalifornia Institute of Technology
InventorGary M. Hathaway
Life of this patent
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Abstract
Methods are provided for determining the position of a post-translationally modified (PTM) amino acid residue such as a phosphorylated amino acid residue or an O-glycosylated amino acid residue in a peptide. Also provided are methods for determining the identity of a PTM amino acid residue in a peptide. In addition, kits for practicing such methods are provided.
Claims27
A method for identifying a position of at least one post-translationally modified (PTM) amino acid residue in at least one peptide, comprising
- a) treating a sample comprising at least one peptide with at least one reagent that can chemically modify a PTM amino acid residue of a peptide, wherein the PTM amino acid residue comprises a phosphorylated amino acid residue, whereby, when a peptide comprises at least one PTM amino acid residue, a peptide comprising at least one chemically modified amino acid residue is generated,
- wherein the peptide is susceptible to cleavage by a proteolytic agent at the position of the chemically modified amino acid residue, and
- wherein the peptide is not susceptible to cleavage by the proteolytic agent at the position of the PTM amino acid residue prior to chemically modifying the PTM amino acid residue;
- b) contacting the peptide comprising the chemically modified amino acid residue with the proteolytic agent, whereby the peptide is cleaved at the position of the chemically modified amino acid residue to produce cleavage products indicative of the position of a PTM amino acid residue in the peptide;
- and
- c) determining the mass of one or more of the cleaved products, thereby identifying the position of at least one PTM amino acid residue in at least one peptide.
A method for identifying a position of at least one post-translationally modified (PTM) amino acid residue in at least one peptide, wherein the PTM amino acid residue comprises phosphoserine, or phosphothreonine, comprising
- a) chemically modifying the PTM amino acid residue of at least one peptide of a sample to produce a lysine analog, thereby generating a peptide comprising at least one lysine analog;
- b) contacting the peptide comprising the lysine analog with a lysine specific proteolytic agent, whereby the peptide is cleaved at the position of the lysine analog to produce cleavage products indicative of the position of a PTM amino acid residue in the peptide;
- and
- c) determining the mass of one or more of the cleaved products, thereby identifying the position of at least one PTM amino acid residue in at least one peptide.
A kit, comprising:
- a) a reagent that effects β-elimination of a phosphoserine, or phosphothreonine, residue of a peptide to produce an amino acid analog Michael acceptor;
- b) a reagent comprising a nucleophile that can interact with the amino acid Michael acceptor to produce a chemically modified amino acid residue in a peptide;
- c) one or more proteolytic agents that can cleave a peptide at the position of the chemically modified amino acid residue formed by β-elimination and Michael addition of phosphoserine, or phosphothreonine residue;
- and
- d) at least one container comprising the reagents and agents.
Dependencies are inferred from the claim text.