Method for assaying protein nitrosylation
View Patent ↗Many of the effects of nitric oxide are mediated by the direct modification of cysteine residues resulting in an adduct called a nitrosothiol. A method to detect proteins which contain nitrosothiols involves several steps. Nitrosylated cysteines are converted to tagged cysteines. Tagged proteins can then be detected, for example, by immunoblotting and/or can be purified by affinity chromatography. The method is applicable to the detection of S-nitrosylated proteins in cell lysates following in vitro S-nitrosylation, as well as to the detection of endogenous S-nitrosothiols in selected protein substrates.
1. A method for screening for potential drugs useful in modulating protein nitrosylation, comprising:
contacting a test sample comprising at least one protein substrate with a test compound;
treating the test sample with an alkylthiolating agent to block free thiol groups on the protein substrate;
reducing nitrosothiol bonds on the protein substrate to form new free thiol groups;
removing the alkylthiolating agent from the test sample;
reacting the new free thiol groups on the protein substrate with a detectably tagged, activated mixed disulfide, thereby forming detectably tagged protein substrate, wherein the detectably tagged protein substrate indicates that the protein substrate in the test sample was nitrosylated;
detecting the detectably tagged protein substrate; and
comparing the amount of detectably tagged protein substrate in the test sample to an amount of detectably tagged protein substrate in a control sample similarly treated but not contacted with the test compound, wherein a test compound which increases or decreases the amount of detectably tagged protein substrate in the test sample relative to the control sample is identified as a modulator of protein nitrosylation.
2. The method of claim 1 wherein the alkylthiolating agent is methyl methanethiosulfonate (MMTS).
3. The method of claim 1 wherein ascorbate is added to the protein substrate during the step of reducing to accelerate the rate of reduction.
4. The method of claim 1 wherein the detectably tagged, activated mixed disulfide is biotinylated aromatic disulfide reagent N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)-propionamide (biotin-HPDP).
5. The method of claim 1 wherein the activated mixed disulfide comprises a radiolabel.
6. The method of claim 1 wherein the activated mixed disulfide comprises biotin.
7. The method of claim 1 wherein the activated mixed disulfide comprises dinitrophenol.
8. The method of claim 1 wherein the activated mixed disulfide comprises a peptide tag.
9. The method of claim 1 wherein the activated mixed disulfide comprises biotin and the biotin is detected using a biotin-specific antibody.
10. The method of claim 1 wherein the protein substrate is detected using an antibody specific for said protein substrate.
11. The method of claim 1 wherein the activated mixed disulfide comprises biotin and the biotin is detected using avidin.
12. The method of claim 1 wherein the activated mixed disulfide comprises biotin and the biotin is detected using streptavidin.
13. The method of claim 1 wherein the test sample is from brain tissue.
14. The method of claim 1 wherein the test sample is from blood vessels.
15. The method of claim 1 wherein the test sample is from macrophages.
16. The method of claim 1 wherein the test sample comprises eNOS.
17. The method of claim 1 wherein the test sample comprises nNOS.
18. The method of claim 1 wherein the test sample comprises iNOS.
19. The method of claim 1 wherein the protein substrate is glyceraldehyde-3 phosphate dehydrogenase.
20. The method of claim 1 wherein the protein substrate is creatine kinase.
21. The method of claim 1 wherein the protein substrate is hexokinase-1.
22. The method of claim 1 wherein the protein substrate is glycogen phosphorylase.
23. The method of claim 1 wherein the protein substrate is NR1 and/or NR2 subunits of NMDA glutamate receptor.
24. The method of claim 1 wherein the protein substrate is isoform 2 or 3 of hyperpolarization-activated cation channel (HAC).
25. The method of claim 1 wherein the protein substrate is α1 or α2 subunit of Na + /K + ATPase.
26. The method of claim 1 wherein the protein substrate is neurofilament H-chain.
27. The method of claim 1 wherein the protein substrate is α or β tubulin.
28. The method of claim 1 wherein the protein substrate is β or γ actin.
29. The method of claim 1 wherein the protein substrate is Retinoblastoma Gene Product, RB.
30. The method of claim 1 wherein the protein substrate is inducible heat shock protein 72.
31. The method of claim 1 wherein the protein substrate is collapsin response mediator protein 1, 2, and/or 4.
32. The method of claim 1 wherein the protein substrate is calbindin.
33. The method of any of claims 11 – 24 wherein an antibody specific for the protein substrate is used in the step of detecting.
34. The method of claim 1 wherein the step of removing involves subjecting the test sample to a spin column.
35. The method of claim 1 wherein the step of removing involves subjecting the test sample to acetone precipitation.
36. The method of claim 1 wherein SDS is added to the test sample to expose hidden thiol groups.