Bioluminescent succinate detection assay

1. A method for detecting or determining the presence or amount of succinate in a sample, the method comprising:

(a) contacting the sample with a first detection reagent to form a first reaction mixture;

(b) contacting the first reaction mixture with a second detection reagent to form a second reaction mixture; and

(c) detecting luminescence in the second reaction mixture, thereby detecting or determining the presence or amount of succinate in the sample;

wherein if succinate is present in the sample, the first detection reagent and the second detection reagent converts the succinate to ATP, wherein:

(i) the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, and acetoacetyl-CoA and the second detection reagent comprises succinyl-CoA ligase (SCS), ADP, a bioluminescent enzyme, and a luciferin substrate;

(ii) the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, acetoacetyl-CoA, succinyl-CoA ligase (SCS), ADP, magnesium chloride, and the second detection reagent comprises a bioluminescent enzyme, and a luciferin substrate; or

(iii) the first detection reagent comprises GDP forming succinyl-CoA ligase (SCS-GDP), GTP, and coenzyme A and the second detection reagent comprises guanylate kinase (GMPK), ADP, a bioluminescent enzyme, and a luciferin substrate.

2. The method of claim 1 , wherein contacting the first reaction mixture with a second detection reagent generates ATP.

3. The method of claim 1 , wherein the sample comprises a cell, a cell lysate, or a tissue sample.

4. The method of claim 1 , wherein the bioluminescent enzyme is luciferase and/or the luciferin substrate is D-luciferin.

5. A method for detecting or determining the presence or amount of a succinate forming enzyme in a sample or the activity of a succinate forming enzyme, the method comprising:

(a) contacting the sample with a peptide, protein, or non-protein substrate, 2-oxoglutarate, Fe(II), and ascorbate to form a succinate reaction mixture, wherein succinate is formed if the sample comprises a succinate forming enzyme;

(b) contacting the succinate reaction mixture with a first detection reagent to form a first reaction mixture, wherein the succinate formed in step (a) is converted to succinyl-CoA;

(c) contacting the first reaction mixture with a second detection reagent to form a second reaction mixture; and

(d) detecting luminescence in the second reaction mixture, thereby detecting or determining the presence or amount of succinate forming enzyme or succinate forming enzyme activity in the sample,

wherein:

(i) the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, and acetoacetyl-CoA and the second detection reagent comprises succinyl-CoA ligase (SCS), ADP, a bioluminescent enzyme, and a luciferin substrate;

(ii) the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, acetoacetyl-CoA, succinyl-CoA ligase (SCS), ADP, magnesium chloride, and the second detection reagent comprises a bioluminescent enzyme, and a luciferin substrate; or

(iii) the first detection reagent comprises GDP forming succinyl-CoA ligase (SCS-GDP), GTP, and coenzyme A and the second detection reagent comprises guanylate kinase (GMPK), ADP, a bioluminescent enzyme, and a luciferin substrate.

6. The method of claim 5 , wherein contacting the first reaction mixture with a second detection reagent generates ATP.

7. The method of claim 5 , wherein the succinate forming enzyme is a 2-oxoglutarate oxygenase.

8. The method of claim 7 , wherein the 2-oxoglutarate oxygenase is a Fe(II)-dependent lysine demethylase or a 2-oxoglutarate-dependent dioxygenase.

9. The method of claim 8 , wherein the Fe(II)-dependent lysine demethylase is a JumonjiC domain-containing histone lysine (JMJC) demethylase.

10. The method of claim 5 , wherein the sample comprises a cell, a cell lysate, or a tissue sample.

11. The method of claim 5 , wherein the bioluminescent enzyme is luciferase and/or the luciferin substrate is D-luciferin.

12. A method for determining whether a compound modulates 2-oxoglutarate oxygenase activity in a sample, the method comprising:

(a) contacting the sample with the compound to form a test sample, wherein the sample comprises a 2-oxoglutarate oxygenase;

(b) contacting the test sample with a peptide, protein, or non-protein substrate, 2-oxoglutarate, Fe(II), and ascorbate to form a succinate reaction mixture;

(c) contacting the succinate reaction mixture with: a first detection reagent to form a first reaction mixture;

(d) contacting the first reaction mixture with a ATP detection reagent or a second detection reagent to form a second reaction mixture;

(e) contacting the second reaction mixture with a ADP-to-ATP conversion/detection reagent to form a third reaction mixture if a second detection reagent is used in step (d);

(f) detecting luminescence in the second reaction mixture of step (d) or the third reaction mixture of step (e); and

(g) comparing the luminescence in the second reaction mixture of step (d) or the third reaction mixture of step (e) to luminescence in a control sample, wherein if the luminescence in the second reaction mixture of step (d) or the third reaction mixture of step (e) is different from the luminescence in a control sample, the compound is identified as being a modulator of 2-oxoglutarate oxygenase activity, wherein:

(i) the first detection reagent comprises: (A) 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, and acetoacetyl-CoA to form a first reaction mixture, whereby the succinate formed in step (a) is converted to succinyl-CoA; or (B) 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, acetoacetyl-CoA, succinyl-CoA ligase (SCS), ADP, magnesium chloride; and the ATP detection reagent comprises: succinyl-CoA ligase (SCS), ADP, a bioluminescent enzyme, and a luciferin substrate; or

(ii) the first detection reagent comprises: GDP forming succinyl-CoA ligase (SCS-GDP), GTP, and coenzyme A; and the ATP detection reagent comprises: guanylate kinase (GMPK), ADP, a bioluminescent enzyme, and a luciferin substrate.