Cell Signaling Technology, Inc.

Cell Singaling Technology, Inc.

John Edward Rush, Ii

Roberto Polakiewicz

Albrecht Moritz

Ailan Guo

Jing Li

Michael Comb

Jing Zhou

Jun-Ming Cai

Kimberly A Lee

Wan Cheung Cheung

Sean Andre Beausoleil

The invention relates to a method for determining the presence of at least one distinct polypeptide in a biological sample comprising contacting the biological sample with a hydrolyzing agent, wherein the hydrolyzing agent is capable of hydrolyzing the distinct polypeptide in a sequence-specific manner such that at least one distinct peptide having a predetermined peptide measured accurate mass would result if the at least one distinct polypeptide were present in the biological sample, to obtain a hydrolyzed sample; bringing the hydrolyzed sample in contact with a substrate comprising at least one immobilized binding partner, wherein the at least one immobilized binding partner is capable of specifically binding the distinct peptide; removing the hydrolyzed sample from the substrate in a manner such that the distinct peptide would remain bound to the immobilized binding partner; contacting the substrate with an elution solution, wherein the distinct peptide would dissociate from the immobilized binding partner into the elution solution; subjecting a portion of the elution solution to liquid chromatography to segregate a plurality of molecules in the portion of the elution solution to obtain sorted molecules; determining the measured accurate mass of at least one sorted molecule present in the elution solution; and determining the presence of the at least one distinct polypeptide in the biological sample when a measured accurate mass of at least one molecule is substantially equal to the predetermined peptide measured accurate mass.

Mass spectrometry analysis of mutant polypeptides in biological samples

There is provided a motif-specific, context-independent antibody that specifically binds a recurring, modified motif consisting of (i) at least one sumoylated lysine residue, and (ii) one or more degenerate amino acids bound by a peptide bond to said sumoylated lysine residue, said antibody specifically binding said motif in a plurality of non-homologous peptides or proteins within an organism in which it recurs. Also provided is a motif-specific, context-independent antibody that specifically binds a recurring, modified motif consisting of (i) a C-terminal aspartic acid residue, and (ii) one or more degenerate amino acids bound by a peptide bond to said C-terminal aspartic acid residue, said antibody specifically binding said motif in a plurality of non-homologous peptides or proteins within an organism in which it recurs.

Motif-specific and context-independent antibodies that specifically bind to a sumoylated lysine-containing residue

The disclosure relates to antibody reagents that specifically bind to peptides carrying a ubiquitin remnant from a digested or chemically treated biological sample. The reagents allow the technician to identify ubiquitinated polypeptides as well as the sites of ubiquitination on them. The reagents are preferably employed in proteomic analysis using mass spectrometry. The antibody reagents specifically bind to the remnant of ubiquitin (i.e., a diglycine modified epsilon amine of lysine) left on a peptide which as been generated by digesting or chemically treating ubiquitinated proteins. The inventive antibody reagents' affinity to the ubiquitin remnant does not depend on the remaining amino acid sequences flanking the modified (i.e., ubiquitinated) lysine, i.e., they are context independent.

Analysis of ubiquitinated polypeptides

In some embodiments, the invention relates to methods for creating a monoclonal antibody that specifically binds to antigen. The method may start from a polyclonal population of antibodies such as a non-specific polyclonal population or a polyclonal population of antibodies that specifically bind to the antigen. The method includes obtaining nucleic acid molecules encoding heavy and light immunoglobulin chains (or variable regions thereof) of multiple immunoglobulins from an animal; obtaining mass spectra information of peptide fragments of a population of polyclonal immunoglobulins that specifically bind to an antigen of choice; comparing and/or correlating the mass spectra information of the peptide fragments of the polyclonal immunoglobulins with predicted mass spectra information of predicted amino acid sequences encoded by the nucleic acid molecules, and then assembling the heavy and light chains to create an antibody (or variable region thereof) that specifically binds to the antigen.

Methods and reagents for creating monoclonal antibodies

Mass spectroscopy analysis of mutant polypeptides in biological samples

Motif-specific and context-independent antibodies that specifically bind to a cleaved caspase motif

The invention relates to antibody reagents that specifically bind to peptides carrying a ubiquitin remnant from a digested or chemically treated biological sample. The reagents allow the technician to identify ubiquitinated polypeptides as well as the sites of ubiquitination on them. The reagents are preferably employed in proteomic analysis using mass spectrometry. The antibody reagents specifically bind to the remnant of ubiquitin (i.e., a diglycine modified epsilon amine of lysine) left on a peptide which as been generated by digesting or chemically treating ubiquitinated proteins. The inventive antibody reagents' affinity to the ubiquitin remnant does not depend on the remaining amino acid sequences flanking the modified (i.e., ubiquitinated) lysine, i.e., they are context independent.

The invention discloses 95 novel phosphorylation sites identified in signal transduction proteins and pathways downstream of the T-cell receptor, and provides phosphorylation-site specific antibodies and heavy-isotope labeled peptides (AQUA peptides) for the selective detection and quantification of these phosphorylated sites/proteins, as well as methods of using the reagents for such purpose. Among the phosphorylation sites identified are sites occurring in the following protein types: Actin Binding proteins, Adaptor/Scaffold proteins, Adhesion proteins, Calcium-binding proteins, Cell Cycle Regulation or Channel proteins, Chaperones, Cofactor proteins, Cytoskeletal proteins, DNA Binding proteins, G protein or GTPase Activating proteins, Ligases, Lipid Kinases and Binding proteins, Oxidoreductases, Protein Kinases, Protein Phosphatases, Receptor proteins, RNA Binding proteins, Transcription Factor/Initiation Complex proteins, Transcription Coactivator/Corepressor proteins, Translation Initiation Complex proteins, Ubitquitin Conjugating System proteins, and Vesicle proteins.

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Beverly, MA, United States of America

John Edward Rush, Ii