Methods of assaying proteins
Methods and systems for identifying a protein within a sample are provided herein. A panel of antibodies are acquired, none of which are specific for a single protein or family of proteins. Additionally, the binding properties of the antibodies in the panel are determined. Further, the protein is iteratively exposed to a panel of antibodies. Additionally, a set of antibodies which bind the protein are determined. The identity of the protein is determined using one or more deconvolution methods based on the known binding properties of the antibodies to match the set of antibodies to a sequence of a protein.
1. A method of identifying individual proteins of a plurality of different proteins, comprising:
(a) providing a plurality of different proteins, individual proteins of the plurality being immobilized at unique spatial addresses, respectively;
(b) iteratively exposing the plurality of immobilized proteins to different labeled monoclonal antibodies or binding fragments thereof, individual labeled monoclonal antibodies or binding fragments thereof of the different labeled monoclonal antibodies or binding fragments thereof binding to two or more different proteins of the plurality of different proteins, and each of the individual labeled monoclonal antibodies or binding fragments thereof having a known degree of binding non-specificity comprising the individual labeled monoclonal antibodies or binding fragments thereof recognizing more than one epitope in the plurality of different proteins;
(c) determining for the unique spatial addresses, respectively:
(i) a set of the labeled monoclonal antibodies or binding fragments thereof that bind to the individual protein at the unique spatial address, and
(ii) a set of the labeled monoclonal antibodies or binding fragments thereof that do not bind to the individual protein at the unique spatial address, thereby determining a pattern of binding of the labeled monoclonal antibodies or binding fragments thereof at the unique spatial addresses, respectively;
(d) identifying individual proteins of the plurality of different proteins by inputting to a Bayesian inference algorithm the known degree of binding non-specificity for each of the individual labeled monoclonal antibodies or binding fragments thereof and the pattern of binding and not binding of the labeled monoclonal antibodies or binding fragments thereof at each of the unique spatial addresses, respectively.
2. The method of claim 1 , wherein the quantity of different proteins identified in step (d) is greater than the quantity of different labeled monoclonal antibodies or binding fragments thereof to which the plurality of immobilized proteins is exposed in step (b).
3. The method of claim 1 , wherein the quantity of different labeled monoclonal antibodies or binding fragments thereof is less than 500.
4. The method of claim 3 , wherein the quantity of different proteins identified in step (d) is at least 1000.
5. The method of claim 1 , wherein the unique spatial addresses are on a substrate surface.
6. The method of claim 1 , wherein the unique spatial addresses comprise beads.
7. The method of claim 1 , wherein the individual proteins of the plurality of different proteins are immobilized via nucleic acids.
8. The method of claim 1 , wherein the labeled monoclonal antibodies or binding fragments thereof comprise fluorescent tags.
9. The method of claim 8 , wherein the fluorescent tags are detected in step (c).
10. The method of claim 1 , wherein the labeled monoclonal antibodies or binding fragments thereof bind the proteins with reproducible specificity.
11. The method of claim 1 , wherein the labeled monoclonal antibodies or binding fragments thereof comprise antibody fragments.
12. A method of identifying individual proteins of a plurality of different proteins, comprising:
(a) providing a plurality of different proteins, individual proteins of the plurality being immobilized at unique spatial addresses, respectively;
(b) iteratively exposing the plurality of immobilized proteins to different aptamers, individual aptamers of the different aptamers binding to two or more different proteins of the plurality of different proteins, and each of the individual aptamers having a known degree of binding non-specificity comprising the individual aptamer recognizing more than one epitope in the plurality of different proteins;
(c) determining for the unique spatial addresses, respectively:
(i) a set of the aptamers that bind to the individual protein at the unique spatial address, and
(ii) a set of the aptamers that do not bind to the individual protein at the unique spatial address, thereby determining a pattern of binding of the aptamers at the unique spatial addresses, respectively;
(d) identifying individual proteins of the plurality of different proteins by inputting to a Bayesian inference algorithm the known degree of binding non-specificity for each of the individual aptamers and the pattern of binding and not binding of the aptamers at each of the unique spatial addresses, respectively.
13. The method of claim 1 , wherein the labeled monoclonal antibodies or binding fragments thereof bind to amino acids comprising post translational modifications.
14. The method of claim 13 , further comprising treating the plurality of different proteins to remove the post translational modifications.
15. The method of claim 1 , further comprising treating the plurality of different proteins to produce shorter polypeptides.
16. The method of claim 1 , wherein the identifying of step (d) comprises determining the most likely identity of individual proteins respectively by solving a satisfaction problem using an algorithm running on a computer system.
17. The method of claim 1 , wherein the identifying of step (d) comprises determining the most likely identity of individual proteins respectively using an algorithm running an expectation maximization approach on a computer system.
18. The method of claim 12 , wherein the aptamers comprise digital aptamers that bind to epitopes consisting essentially of 3 consecutive amino acids.
19. The method of claim 12 , wherein the aptamers comprise digital aptamers that bind to epitopes consisting essentially of 4 consecutive amino acids.
20. The method of claim 1 , wherein the plurality of different proteins comprises more than 1000 different proteins.
21. The method of claim 1 , wherein the different labeled monoclonal antibodies or binding fragments thereof comprise an affinity reagent that recognizes several different epitopes.
22. The method of claim 1 , wherein the different labeled monoclonal antibodies or binding fragments thereof comprise a labeled monoclonal antibody or binding fragment thereof that recognizes a family of one or more epitopes that are present in more than one of the different proteins.
23. The method of claim 1 , wherein the labeled monoclonal antibodies or binding fragments thereof bind to an amino acid motif consisting essentially of 2 consecutive amino acids.
24. The method of claim 1 , wherein the known degree of binding non-specificity for each of the individual labeled monoclonal antibodies or binding fragments thereof and the pattern of binding of the labeled monoclonal antibodies or binding fragments thereof at the unique spatial addresses are input to a Bayesian inference algorithm to identify the individual proteins.
25. The method of claim 1 , wherein the plurality of different proteins comprises a sample derived from a biological source and wherein high abundance proteins are removed from the sample prior to immobilizing the plurality of different proteins at the unique spatial addresses.