Abstract: A method is provided for producing motif-specific, context-independent antibodies which recognize a plurality of peptides or proteins within a genome that contain the same motif. The method includes the step of immunizing a host with a degenerate peptide library antigen featuring (i) a fixed target motif containing one or more invariant amino acids including at least one modified amino acid, and (ii) a plurality of degenerate amino adds flanking the motif. Motif-specific, context-independent antibodies produced by the disclosed method are also provided. The method encompasses motifs consisting of a single modified amino acid, as well as short motifs comprising multiple invariant amino acids including one or more modified amino acids, such as all or part of kinase consensus substrate motifs, protein-protein binding motifs, or other cell signaling motifs. Methods of using the antibodies, e.g. for genome-wide profiling, are also provided.

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.