Immunoaffinity isolation of modified peptides from complex mixtures
First Claim
1. A method of creating a record, the method comprising:
- (a) obtaining a digested proteinaceous preparation comprising a complex mixture of peptides comprising naturally-occurring post-translationally modified peptides from two or more different proteins;
(b) contacting said digested proteinaceous preparation with at least one immobilized post-translational modification-specific antibody;
(c) isolating a population of naturally-occurring post-translationally modified peptides specifically bound by said immobilized modification-specific antibody;
(d) characterizing said population of modified peptides by mass spectrometry to identify one or more modified peptides of the population; and
(e) creating a record comprising information relating to the one or more modified peptides of the population.
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Abstract
The invention provides methods for isolating a modified peptide from a complex mixture of peptides, the method comprising the steps of: (a) obtaining a proteinaceous preparation from an organism, wherein the preparation comprises modified peptides from two or more different proteins; (b) contacting the preparation with at least one immobilized modification-specific antibody; and (c) isolating at least one modified peptide specifically bound by the immobilized modification-specific antibody in step (b). The method may further comprise the step of (d) characterizing the modified peptide isolated in step (c) by mass spectrometry (MS), tandem mass spectrometry (MS-MS), and/or MS3 analysis, or the step of (e) utilizing a search program to substantially match the spectra obtained for the modified peptide during the characterization of step (d) with the spectra for a known peptide sequence, thereby identifying the parent protein(s) of the modified peptide. Also provided are an immunoaffinity isolation device comprising a modification-specific antibody, and antibodies against novel UFD1 and PTN6 phosphorylation sites.
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Citations
26 Claims
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1. A method of creating a record, the method comprising:
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(a) obtaining a digested proteinaceous preparation comprising a complex mixture of peptides comprising naturally-occurring post-translationally modified peptides from two or more different proteins; (b) contacting said digested proteinaceous preparation with at least one immobilized post-translational modification-specific antibody; (c) isolating a population of naturally-occurring post-translationally modified peptides specifically bound by said immobilized modification-specific antibody; (d) characterizing said population of modified peptides by mass spectrometry to identify one or more modified peptides of the population; and (e) creating a record comprising information relating to the one or more modified peptides of the population. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A method of creating a record, the method comprising:
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(a) obtaining a digested proteinaceous preparation comprising a complex mixture of peptides comprising naturally-occurring post-translationally modified peptides from two or more different proteins and fractionating said digested proteinaceous preparation by reversed-phased chromatography to produce a fractionated proteinaceous preparation; (b) contacting said fractionated proteinaceous preparation with at least one immobilized post-translational modification-specific antibody; (c) isolating a population of naturally-occurring post-translationally modified peptides specifically bound by said immobilized modification-specific antibody; (d) characterizing said population of modified peptides by mass spectrometry to identify one or more modified peptides of the population; and (e) creating a record comprising information relating to the one or more modified peptides of the population. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
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Specification