Methods and compositions for detecting the activation state of multiple proteins in single cells
View Patent ↗The invention provides methods and compositions for simultaneously detecting the activation state of a plurality of proteins in single cells using flow cytometry. The invention further provides methods and compositions of screening for bioactive agents capable of coordinately modulating the activity of a plurality of proteins in single cells. The methods and compositions can be used to determine the protein activation profile of a cell for predicting or diagnosing a disease state, and for monitoring treatment of a disease state.
1. A method of detecting the activation state of single cells, said method comprising the steps of:
a) providing a cell comprising a first and a second activatable protein, wherein said first and second activatable proteins are different proteins that each have at least an activated isoform, and a non-activated isoform;
b) contacting said cell with at least two activation state-specific antibodies,
wherein a first of said at least two activation state-specific antibodies is specific for said activated isoform of said first activatable protein; and a second of said at least two activation state-specific antibodies is specific for said activated isoform of said second activatable protein, wherein said two activation state-specific antibodies are distinguishably detectable; and
c) detecting a presence or absence of binding of said first and said second activation state-specific antibodies to their corresponding activated isoform of said first and second activatable proteins in said cell, wherein said presence or absence of binding of said first activation state-specific antibody is indicative of the activation state of said first activatable protein, and said presence or absence of binding of said second activation state-specific antibody is indicative of the activation state of said second activatable protein.
2. The method according to claim 1 , wherein step a) further comprises exposing said cell to a stimulus.
3. The method according to claim 2 , wherein said stimulus is an activating agent.
4. The method according to claim 2 , wherein said stimulus is an inhibitor.
5. The method according to claim 2 , wherein said stimulus is selected from the group consisting of cytokines, interleukins, hormones, bioactive agents, receptor ligands, and therapeutic agents.
6. The method according to claim 1 , wherein said first state-specific antibody and said second state-specific antibody are distinguishably labeled.
7. The method according to claim 1 , wherein said first activatable protein is a kinase.
8. The method according to claim 1 , wherein said first activatable protein is a caspase.
9. The method according to claim 1 , wherein said first activatable protein is a first kinase and said second activatable protein is a second kinase.
10. The method according to claim 9 , wherein the activated state of said first kinase is a first phosphorylated kinase, and the activated state of said second kinase is a second phosphorylated kinase.
11. The method according to claim 10 , wherein said first activation state-specific antibody is specific for said first phosphorylated kinase, and said second activation state-specific antibody is specific for said second phorphorylated kinase.
12. The method according to claim 1 , wherein said first activatable protein is a first caspase and said second activatable protein is a second caspase.
13. The method according to claim 12 , wherein the activated state of said first caspase is a cleaved product of a first pro-caspase, and the activated state of said second caspase is a cleaved product of a second pro-caspase.
14. The method according to claim 1 , wherein the activated state of either the first or second activatable protein is created through protein cleavage.
15. The method according to claim 1 , wherein the activated state of either the first or second activatable protein is created through a covalent addition, structural change, or conformational change.
16. The method according to claim 15 , wherein said conformational change causes said activated state of either the first or second activatable protein to present at least one epitope that is not present in a non-activated state of either the first or second activatable protein.
17. The method according to claim 1 , wherein the activated state of either the first or second activatable protein is a phosphorylated or a cleaved product of either the first or second activatable protein.
18. The method according to claim 1 , wherein the activated state of either the first or second activatable protein is created through binding to a third protein or protein complex.
19. The method according to claim 1 , wherein said first activatable protein is a protein within the MAPK, AKT, STAT, NFkB, PKC or WNT signaling pathways and said second activatable protein is a protein within the MAPK, AKT, STAT, NFkB, PKC or WNT signaling pathways.
20. The method according to claim 1 , wherein said first activatable protein and second activatable protein are independently selected from the group consisting of AKT, PI3K, MAPK, p44/42 MAP kinase, TYK2, p38 MAP kinase, PKC, PKA, SAPK, ELK, JNK, cJun, RAS, Raf, MEK 1/2, MEK 3/6, MEK 4/7, ZAP-70, LAT, SRC, LCK, ERK 1/2, Rsk 1, PYK2, SYK, PDK1, GSK3, FKHR, AFX, PLCγ, PLCy, NF-kB, FAK, CREB, αIIIβ3, FcεRI, BAD, p70S6K, STAT1, STAT2, STAT3, STAT5 and STAT6.
21. The method according to claim 20 , wherein said first activatable protein and second activatable protein are independently selected from the group consisting of RAF, ERK 1/2, MEK 1/2, p38, JNK, AKT, PKA substrates, PKC, PLCγ, STAT1, STAT2, STAT3, STAT5 and STAT6.
22. The method according to claim 1 , wherein said step c) is performed with a detector selected from the group consisting of: flow cytometer; microscope having multiple channels of fluorescence; a plate reader configured to detect fluorescence, ultraviolet and/or visible light, fluorescence resonance energy transfer (FRET), luminescence, quenching, two-photon excitation, and/or intensity redistribution; charge-coupled device (CCD) camera configured to capture and transform data and images into quantifiable formats; high performance liquid chromatography (HPLC); and mass spectrometer.
23. The method according to claim 22 , wherein said step c) is performed with a flow cytometer.
24. The method according to claim 1 , wherein said method further comprises sorting said single cell based on said activation state of said first activatable protein, and said activation state of said second activatable protein.
25. The method according to claim 1 , wherein step a) further comprises permeabilizing said cell.
26. The method according to claim 1 , wherein step a) further comprises fixing said cell.
27. The method according to claim 1 , wherein said cell is a mammalian cell.
28. The method according to claim 1 , wherein said first activation state-state specific antibody and said second activation state-state specific antibody are independently selected from the group consisting of anti-phospho-AKT Ser473, anti-phospho-p44/42 MAP kinase (Thr202/Tyr204), anti-phospho-TYK2 (Tyr1054/1055), anti-phospho-p38 MAP kinase (Thr180/Tyr182), phospho-PKC-PAN substrate antibody, phospho-PKA-substrate antibody, anti-phospho-SAPK/JNK (Thr183/Tyr185), anti-phospho-tyrosine (P-tyr-100), anti-p44/42 MAPK, anti-phospho-MEK1/2 (Ser217/221), anti-phospho-p90RSK (Ser381), anti-p38 MAPK, anti-JNK/SAPK, anti-phospho-AKT-Thr308, anti-phospho-Raf1 (Ser259), anti-phosphoElk-1 (Ser383), anti-phospho-CREB (Ser133), anti-phosphoSEK1/MKK4 (Thr261), anti-phospho-Jun (Ser 63), anti-phosphoMKK3/MKK6 (Ser189/207), anti-AKT, anti-phospho FKHR, anti-FKHR, anti-phospho-Gsk3 alp21, anti-pAFX, anti-PARP, anti-BAD, anti-BADser112, anti-BADser136, anti-phospho-BADser155, anti-p27, anti-p21, anti-cFLIP, antiMYC, anti-p53, anti-NFKB, anti-lkkα, anti-lkkβ, anti-phospho-tyrosine, anti-phospho-threonine, anti-STAT1, anti-STAT2, anti-STAT3, anti-STAT5, anti-STAT6, anti-PIP2, and anti-PIP3.
29. A method of detecting the activation state of single cells, said method comprising the steps of:
a) providing a cell comprising a first and a second activatable protein, wherein said first and second activatable proteins are different proteins that each have at least an activated isoform and a non-activated isoform;
b) contacting said cell with at least two activation state-specific binding agents, wherein a first of said at least two activation state-specific binding agents is specific for said activated isoform of said first activatable protein and a second of said at least two activation state-specific binding agents is specific for said activated isoform of said second activatable protein, wherein said two activation state-specific binding agents are distinguishably detectable; and
c) detecting a presence or absence of binding of said first and said second activation state-specific binding agents to their corresponding activated isoform of said first and second activatable proteins in said single cell, wherein said presence or absence of binding of said first activation state-specific binding agent is indicative of the activation state of said first activatable protein, and said presence or absence of binding of said second activation state-specific binding agent is indicative of the activation state of said second activatable protein.
30. The method according to claim 29 , wherein said first activatable protein is a first kinase and said second activatable protein is a second kinase.
31. The method according to claim 30 , wherein the activated isoform of said first kinase is a first phosphorylated kinase, and the activated isoform of said second kinase is a second phosphorylated kinase.
32. The method according to claim 30 , wherein said first activatable protein is a protein within the MAPK, AKT, STAT, NFkB, PKC or WNT signaling pathways and said second activatable protein is a protein within the MAPK, AKT, STAT, NFkB, PKC or WNT signaling pathways.