Immunoaffinity isolation of modified peptides from complex mixtures
First Claim
1. A method for isolating a target population of naturally-occurring post-translatiorially modified peptides from a complex mixture of peptides, said method comprising the steps of:
- (a) obtaining a digested proteinaccous preparation from an organism, wherein said digested proteinaccous preparation comprises a complex mixture of peptides comprising naturally-occurring post-translationally modified peptides from two or more different proteins and further fractionating said post-translationally modified peptides in said digested proteinaceous preparation by reversed-phased chromatography to produce a fractionated proteinaceous preparation;
(b) contacting said fractionated proteinaccous preparation with at least one immobilized post-translational modification-specific antibody; and
(c) isolating said target of naturally-occurring post-translationally modified peptides specifically bound by said immobilized inodification-specific antibody in step (b).
1 Assignment
0 Petitions
Accused Products
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.
-
Citations
43 Claims
-
1. A method for isolating a target population of naturally-occurring post-translatiorially modified peptides from a complex mixture of peptides, said method comprising the steps of:
-
(a) obtaining a digested proteinaccous preparation from an organism, wherein said digested proteinaccous preparation comprises a complex mixture of peptides comprising naturally-occurring post-translationally modified peptides from two or more different proteins and further fractionating said post-translationally modified peptides in said digested proteinaceous preparation by reversed-phased chromatography to produce a fractionated proteinaceous preparation; (b) contacting said fractionated proteinaccous preparation with at least one immobilized post-translational modification-specific antibody; and (c) isolating said target of naturally-occurring post-translationally modified peptides specifically bound by said immobilized inodification-specific antibody in step (b). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33)
-
-
34. A method for isolating a target population of phosphopeptides from a complex mixture of peptides, said method comprising the steps of:
-
(a) obtaining a digested proteinaceous preparation from an organism, wherein said digested proteinaceous preparation comprises a complex mixture of peptides comprising phosphopeptides from two or more different proteins; (b) fractionating phosphopeptides in said digested proteinaceous preparation by reversed-phased chromatography to produce a fractionated proteinaceous preparation; (c) contacting said fractionated proteinaccous preparation with at least one immobilized motif-specific, context-independent antibody that binds a motif comprising at least one phosphorylated amino acid; (d) isolating said target population of phosphopeptides specifically bound by said immobilized antibody in step (c); and (e) characterizing said population of phosphopeptides isolated in step (d) by mass spectrometry (MS), tandem mass spectrometry (MS—
MS), and/or MS3 analysis. - View Dependent Claims (35, 36, 37, 38, 39, 40, 41, 42, 43)
-
Specification