Method for analyzing proteins
View Patent ↗A method for analyzing proteins makes use of an array of first capture molecules which are specific for peptide epitopes. The proteins to be analyzed or a protein mixture containing the proteins to be analyzed is degraded to peptide fragments corresponding to the peptide epitopes, after which the array of capture molecules is incubated with the peptide fragments. The peptide fragments bound to the capture molecules are then detected.
1. A method for detecting plural different proteins in a protein mixture, comprising:
(1) incubating immobilized first capture molecules with denatured linear peptide fragments of the plural different proteins;
wherein the first capture molecules are specific for individual peptide epitopes present in the linear peptide fragments;
wherein the incubation occurs under conditions whereby the first capture molecules specifically bind to and thereby form complexes with the linear peptide fragments;
(2) detecting the linear peptide fragments bound to the immobilized first capture molecules; and
(3) identifying the linear peptide fragments, and thus the proteins that correspond to the linear peptide fragments, on the basis of their binding to the first capture molecules.
2. The method of claim 1 , further comprising washing away unbound peptide fragments after the incubating step.
3. The method of claim 1 , wherein each of the first capture molecules is selected from the group consisting of: an antibody, an antibody fragment, a peptide aptamer and another capture molecule which can be prepared recombinantly by mutation of binding domains and by chemical synthesis.
4. The method of claim 3 , wherein each of the first capture molecules is an antibody or an antibody fragment.
5. The method of claim 4 , wherein each of the first capture molecules is an antibody.
6. The method of claim 1 , wherein the individual peptide epitopes are selected from all potential peptide epitopes of the proteins.
7. The method of claim 6 , wherein the potential peptide epitopes are derived from known amino acid sequences of the proteins.
8. The method of claim 7 , wherein sequence sections which are retained after fragmentation of the proteins are selected as potential peptide epitopes.
9. The method of claim 6 , wherein the potential peptide epitopes are derived from known nucleic acid sequences encoding the proteins.
10. The method of claim 6 , wherein sequence sections which are retained after fragmentation of the proteins are selected as potential peptide epitopes.
11. The method as claimed in claim 1 , wherein the first capture molecules are generated to be specific for the individual peptide epitopes, which are provided with post-translational modifications.
12. The method as claimed in claim 1 , wherein the individual peptide epitopes differ by at least one amino acid compared to a potential peptide epitope of said protein.
13. The method as claimed in claim 1 , wherein the binding specificity of the first capture molecules is established by binding assays using peptides which have been varied in individual amino acids or their length.
14. The method of claim 1 , further comprising contacting said complexes with second capture molecules.
15. The method of claim 14 , wherein the second capture molecules are labeled.
16. The method of claim 14 , wherein the second capture molecules are selected from the group consisting of antibodies, antibody fragments, and peptide aptamers.
17. The method of claim 16 , wherein each of the second capture molecules is an antibody or an antibody fragment.
18. The method of claim 17 , wherein each of the second capture molecules is an antibody.
19. The method of claim 14 , wherein the binding specificity of each of the second capture molecules is established by binding assays using peptides which have been varied in individual amino acids or their length.
20. The method of claim 1 , wherein said individual peptide epitopes used to generate said first capture molecules differ from the corresponding linear peptide fragments by at least one amino acid.
21. The method of claim 1 , wherein said second capture molecules are raised against one of said linear peptide fragments specifically bound to the first capture molecules.