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 assaying 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 affinity reagents, individual affinity reagents of the different affinity reagents binding to two or more different proteins of the plurality of different proteins, and each of the individual affinity reagents having a known degree of binding non-specificity for the plurality of different proteins;
(c) determining for the unique spatial addresses, respectively:
(i) a set of the affinity reagents that bind to the individual protein at the unique spatial address, and
(ii) a set of the affinity reagents that do not bind to the individual protein at the unique spatial address,
thereby determining a pattern of binding of the affinity reagents at the unique spatial addresses, respectively; and
(d) identifying individual proteins of the plurality of different proteins using the known degree of binding non-specificity of the affinity reagents and the pattern of binding of the affinity reagents at the unique spatial addresses, respectively,
wherein identifying the individual proteins comprises determining the most likely identity of the individual proteins using a Bayesian inference algorithm.
2. The method of claim 1 , wherein the quantity of different proteins identified in step (d) is greater than the quantity of different affinity reagents to which the plurality of immobilized proteins is exposed in step (b).
3. The method of claim 1 , wherein the quantity of different affinity reagents 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 affinity reagents comprise fluorescent tags that are detected in step (c).
9. The method of claim 1 , wherein the affinity reagents comprise monoclonal antibodies.
10. The method of claim 1 , wherein the affinity reagents comprise antibody fragments.
11. The method of claim 1 , wherein the affinity reagents comprise aptamers.
12. The method of claim 1 , wherein the affinity reagents bind to amino acids comprising post translational modifications.
13. The method of claim 12 , further comprising treating the plurality of different proteins to remove the post translational modifications.
14. 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.
15. 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.
16. The method of claim 1 , wherein the identifying of step (d) comprises determining the most likely identity of individual proteins respectively using a machine learning algorithm.
17. The method of claim 1 , wherein the different affinity reagents comprise an affinity reagent that recognizes several different epitopes.
18. The method of claim 1 , wherein the different affinity reagents comprise an affinity reagent that recognizes a family of one or more epitopes that are present in more than one of the different proteins.
19. 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.