Methods for identifying protein-protein interactions
The invention generally relates to methods for identifying protein-protein interactions. In certain aspects, methods of the invention involve conducting a protein-fragment complementation assay on a sample to form a protein-protein complex between two proteins in the sample that only transiently interact under physiological conditions, separating the complexes from the sample, and analyzing a protein of the complex using a mass spectrometry technique.
1. A method for analyzing two transiently interacting proteins in a sample, wherein a first protein is fused with a first fragment of a reporter protein and a second protein is fused with a second fragment of said reporter protein, the method comprising:
conducting a protein-fragment complementation assay on the sample to form a protein-protein complex transiently under physiological conditions;
separating the complex from the sample using a moiety that specifically binds a fused form of the reporter protein but not either of the first and second proteins individually, and applying a washing condition to the sample that removes the first and second proteins in their free form while preserving the complex; and
quantitatively analyzing at least one protein of the complex using a mass spectrometry technique.
2. The method according to claim 1 , wherein the reporter protein is a bimolecular fluorescence protein.
3. The method according to claim 1 , wherein separating comprises:
exposing the complexes to one or more solid supports, each solid support comprising the moiety that specifically binds the fused form of the reporter protein; and
washing away remaining components of the sample.
4. The method according to claim 3 , wherein the moiety is an antibody that specifically binds the fused form of the reporter protein.
5. The method according to claim 4 , wherein the antibody is selected from the group consisting of an anti-fluorescence protein antibody, a nanobody and a variable single domain antibody fragment (V H H).
6. The method according to claim 3 , wherein the fused form of the reporter protein comprises a first member of a binding pair, and the moiety is a second member of the binding pair.
7. The method according to claim 3 , wherein the solid supports are beads.
8. The method according to claim 3 , wherein prior to the analyzing step, the method further comprises:
eluting the complexes from the solid supports; and
digesting the proteins to from peptides.
9. The method according to claim 1 , wherein one protein of the complex is a kinase.
10. A method for analyzing two transiently interacting proteins in a sample, wherein a first protein is fused with a first fragment of a reporter protein and a second protein is fused with a second fragment of said reporter protein, the method comprising:
conducting a protein-fragment complementation assay on the sample to form a protein-protein complex transiently under physiological conditions, and the assay is conducted in the presence of a competitor;
separating the complex from the sample using a moiety that specifically binds a fused form of the reporter protein but not either of the first and second proteins individually, and applying a washing condition to the sample that removes the first and second proteins in their free form while preserving the complex; and
quantitatively analyzing a protein of the complex using a mass spectrometry technique.
11. The method according to claim 10 , wherein the reporter protein is a bimolecular fluorescence protein.
12. The method according to claim 10 , wherein separating comprises:
exposing the complexes to one or more solid supports, each solid support comprising the moiety that specifically binds the fused form of the reporter protein; and
washing away remaining components of the sample.
13. The method according to claim 12 , wherein the moiety is an antibody that specifically binds the fused form of the reporter protein.
14. The method according to claim 13 , wherein the antibody is selected from the group consisting of an anti-fluorescence protein antibody, a nanobody and a variable single domain antibody fragment (VHH).
15. The method according to claim 12 , wherein the fused form of the reporter protein comprises a first member of a binding pair, and the moiety is a second member of the binding pair.
16. The method according to claim 12 , wherein the solid supports are beads.
17. The method according to claim 12 , wherein prior to the analyzing step, the method further comprises:
eluting the complexes from the solid supports; and
digesting the proteins to from peptides.
18. The method according to claim 10 , wherein one protein of the complex is a kinase.
19. The method according to claim 1 , wherein the analyzing step provides an amino acid modification status of at least one protein in the complex.