Multiplex protein fractionation
AssigneeTethys Bioscience, Inc.
InventorsMichael S. Urdea, Michael P. McKenna, Scott Eastman
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Abstract
The invention provides methods, compositions, and kits for multiplex fractionation of proteins in a sample. Protein-binding molecules, such as small epitope antibodies or small epitope aptamers, are used for multiplex fractionation of proteins in a protein containing sample. Detection of fractionated proteins may be used for characterization of proteins in a sample in applications such as expression profiling, identification and/or quantification of proteins in a sample, and identification or detection of biomarkers.
Claims45
A method for characterizing protein in a sample that comprises a mixture of proteins, said method comprising:
- (a) fractionating protein in said sample into fractions with a plurality of first protein-binding molecules;
- (b) contacting protein in at least one of said fractions with a plurality of second protein-binding molecules;
- and
- (c) detecting protein bound to said second protein-binding molecules.
A method for characterizing protein in a sample that comprises a mixture of proteins, said method comprising:
- (a) contacting protein in said sample with a protein cleaving agent, wherein said protein cleaving agent cleaves at a cleavage site to produce a set of polypeptide fragments comprising a C-terminal amino acid at said cleavage site;
- and wherein said polypeptide fragments comprise internal small epitopes;
- (b) fractionating said polypeptide fragments into fractions with a plurality of first protein-binding molecules wherein said first protein-binding molecules bind to small epitopes comprising said C-terminal amino acid;
- (c) contacting polypeptide fragments in at least one of said fractions with a plurality of second protein-binding molecules wherein said second protein-binding molecules bind to said internal epitopes;
- and
- (d) detecting protein bound to said second protein-binding molecules.
A method for characterizing protein in a sample that comprises a mixture of proteins, said method comprising:
- (a) fractionating protein in said sample into fractions with a plurality of first small epitope antibodies, thereby forming first small epitope antibody-protein complexes;
- (b) contacting protein in at least one of said fractions with a plurality of second small epitope antibodies, thereby forming second small epitope antibody-protein complexes;
- and
- (c) detecting protein bound to said second small epitope antibodies.
A method of identifying a protein in a sample, said method comprising:
- (a) contacting protein in a sample with a first small epitope antibody that recognizes a first small epitope, thereby forming a first small epitope antibody-protein complex;
- (b) contacting protein in the first small epitope antibody-protein complex with a second small epitope antibody that recognizes a second small epitope, thereby forming a second small epitope antibody-protein complex, wherein said second small epitope antibody-protein complex comprises protein comprising said first small epitope and said second small epitope;
- and
- (c) reviewing known amino acid sequences of proteins from the organism from which the sample was derived to identify one or more proteins comprising said first and second small epitopes, whereby protein in said second small epitope antibody-protein complex is identified as said one or more proteins.
A method for characterizing protein in a sample that comprises a mixture of proteins, said method comprising:
- (a) contacting protein in the sample with a protein cleaving agent wherein said protein cleaving agent cleaves at a cleavage site to produce a set of polypeptide fragments comprising a C-terminal amino acid at said cleavage site;
- (b) contacting protein with a plurality of protein-binding molecules wherein said protein-binding molecules bind to small epitopes comprising said C-terminal amino acid;
- (c) detecting unbound protein.
Dependencies are inferred from the claim text.