Multiplex protein fractionation
View Patent ↗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.
1. 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.
2. A method according to claim 1 , wherein each of said second protein-binding molecules comprises a unique detectable label, and wherein the method comprises detecting the unique detectable labels.
3. A method according to claim 1 , wherein said small epitope comprising a C-terminal amino acid consists of 3 to 5 amino acids.
4. A method according to claim 1 , wherein said internal epitopes consist of 3 to 5 amino acids.
5. 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.
6. A method according to claim 5 , wherein said protein bound to said protein-binding molecules is separated from said unbound protein prior to detection.
7. A method according to claim 5 , wherein said protein comprises a detectable label, and wherein the method comprises detecting the label in said unbound protein.
8. A method according to claim 5 , wherein said protein-binding molecules comprise small epitope antibodies.
9. A method according to claim 5 , wherein said protein-binding molecules comprise small epitope aptamers.