Automated systems and methods for analysis of protein post-translational modification
First Claim
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1. An automated method for identifying modified 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 post-translationally modified with a moiety of interest;
(ii) processing the protein samples to chemically modify at least one of the C-terminal carboxyl, the N-terminal amine and amino acid side chains of proteins in the protein samples so as to increase the specificity of the affinity capture reagent for those proteins which have been post-translationally modified with the moiety of interest;
(iii) isolating proteins post-translationally with the moiety of interest from the proteins samples using the affinity capture reagent; and
, (iv) determining the identity of isolated proteins by mass spectroscopy;
wherein the method is performed using an apparatus comprising an autosampler, an affinity-capture apparatus and an ion source.
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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 modifications, such as phosphorylation.
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Citations
31 Claims
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1. An automated method for identifying modified amino acids within a protein, comprising:
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(i) providing one or more protein samples and an affinity capture reagent for isolating, from the protein samples, those proteins which have been post-translationally modified with a moiety of interest;
(ii) processing the protein samples to chemically modify at least one of the C-terminal carboxyl, the N-terminal amine and amino acid side chains of proteins in the protein samples so as to increase the specificity of the affinity capture reagent for those proteins which have been post-translationally modified with the moiety of interest;
(iii) isolating proteins post-translationally with the moiety of interest from the proteins samples using the affinity capture reagent; and
,(iv) determining the identity of isolated proteins by mass spectroscopy;
wherein the method is performed using an apparatus comprising an autosampler, an affinity-capture apparatus and an ion source. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 23, 24, 25, 26, 27, 30, 31)
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16. A method for analyzing a phosphoproteome, comprising:
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(i) for a protein sample, chemically modify the side chains of glutamic acid and aspartic acid residues to neutral derivatives;
(ii) isolating phosphorylated proteins from the protein sample by immobilized metal affinity chromatography;
(iii) determining the identity of isolated proteins by mass spectroscopy. - View Dependent Claims (17, 18, 19, 20, 21, 22, 28, 29)
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Specification