Cell Signaling Technology, Inc.

Other

Yi Tan

Michael Comb

Hui Zhang

Xiangming Zha

Peter Hornbeck

John B Rush

John Rush

Michael J Comb

A method is provided for producing motif-specific, context-independent antibodies that recognize a plurality of peptides or proteins within a genome that contain the same post-translationally modified 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 acids 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.

Production of motif-specific and context-independent antibodies using peptide libraries as antigens

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 MSanalysis, 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.

Immunoaffinity isolation of modified peptides from complex mixtures

The present invention is related to a method for producing motif-specific, context-independent antibodies which are specific to at least one modified amino acid residue in the context of variable surrounding amino acid or peptide sequences. The method is particularly useful in producing antibodies which recognize phosphorylated serine, threonine, and tyrosine, or acetylated lysine, as well as other modified amino acids-containing motifs of one or more amino acids.

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&#x2014;MS), and/or MSanalysis, 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.

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.

A class of motif-specific, context-independent antibodies that bind conserved signal transduction motifs, such as kinase consensus substrate motifs and protein-protein binding motifs, containing one or more modified amino acids, such as a phosphorylated amino acid, is provided. The antibodies bind a plurality of peptides or proteins that contain the modified motif. Context-independent antibodies specific for single modified residues, such as phosphothreonine, are also provided. Methods for producing and using such antibodies are provided.

The present invention is related to a method for producing motif-specific, context-independent antibodies which are specific to at least one modified fixed amino acid residue in the context of variable surrounding amino acid or peptide sequences. The method is particularly useful in producing antibodies which recognize phosphorylated serine, threonine, and tyrosine, or acetylated lysine, as well as other modified amino acid-containing motifs of one or more amino acids.

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Lynnfield, MA, United States of America

Yi Tan