Systems and methods for the analysis of protein phosphorylation
AssigneeUniversity of Virginia Patent Foundation
InventorsDonald F. Hunt, Forest M. White, Scott Ficarro
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Abstract
Methods and systems of applying mass spectrometry to the analysis of peptides and amino acids, especially in the proteome setting. More particularly, the invention relates to a mass spectrometry-based method for detection of amino acid phosphorylation.
Claims35
A method for identifying phosphorylated amino acids within a protein, comprising:
- (i) providing one or more protein samples and an affinity capture reagent for isolating, from the protein samples, those proteins which have been phosphorylated;
- (ii) processing the protein samples to chemically modify at least one of the C-terminal carboxyl and amino acid side chains of proteins in the protein samples;
- (iii) isolating phosphorylated proteins from the proteins samples using the affinity capture reagent;
- (iv) determining the identity of isolated proteins by mass spectroscopy.
A method for analyzing a phosphoproteome, comprising:
- (i) providing a protein sample;
- (ii) chemically modifying the side chains of glutamic acid and aspartic acid residues to neutral ester derivatives;
- (iii) isolating phosphorylated proteins from the protein sample by immobilized metal affinity chromatography;
- (iv) determining the identity of isolated proteins by mass spectroscopy.
An improved method of using IMAC to isolate phosphoproteins from a complex mixture, the method comprising the step of modifying carboxylic acid groups present in the mixture to neutral carboxy groups prior to subjecting the mixture to IMAC purification.
A method for identifying phosphorylated amino acids within a peptide, comprising;
- (i) providing one or more peptide-containing samples and an affinity capture reagent for isolating, from the samples, those peptides which have been phosphorylated;
- (ii) processing the samples to chemically modify at least one of the C-terminal carboxyl and amino acid side chains of peptides in the samples;
- (iii) isolating phosphorylated peptides from the samples using the affinity capture reagent;
- (iv) determining the identity of the peptides by mass spectroscopy.
Dependencies are inferred from the claim text.