Assays for detecting modified compounds
Provided are methods and compositions which are useful for separating, isolating, detecting, and quantifying compounds of interest which have been modified chemically, enzymatically or catalytically from other compounds which have not been so modified. The modifications may take the form of functional groups which are gained, lost or retained by the compounds of interest.
1. A method for isolating a compound which has gained at least one functional group, the method comprising
(a) providing:
(i) a compound which can gain a functional group, the compound being directly covalently bound to at least one labeled non-radioactive signaling moiety;
(ii) a chemical, enzymatic or catalytic source of the functional group which can be gained by the compound; and
(iii) means to separate the compound which has gained the functional group from the compound which has not gained the functional group;
(b) forming a mixture comprising the labeled compound and the source of the functional group, and incubating the mixture under conditions suitable for the compound to gain the functional group;
(c) separating and thereby isolating any compound which has gained the functional group from any compound which has not gained the functional group by precipitating said compound which has gained the functional group from the mixture; and
(d) resolubilizing said compound which has gained the functional group.
2. The method of claim 1 , further comprising detecting or quantifying the non-radioactive signaling moiety in said resolubilized compound having the functional group, to detect or quantify any compound which has gained the functional group.
3. The method of claim 1 , wherein the compound comprises a macromolecule or a small biological compound.
4. The method of claim 3 , wherein the macromolecule or small biological compound comprises a nucleic acid, a protein, a sugar, a polysaccharide, a lipid, a glycoprotein, a glycolipid, or a lipoprotein.
5. The method of claim 3 , wherein the macromolecule or small biological compound comprises an oligomer or a polymer.
6. The method of claim 5 , wherein the oligomer or polymer comprises a nucleic acid, an abasic nucleic acid, a peptide nucleic acid, an oligo- or polypeptide, a protein, an oligosaccharide, a polysaccharide or an organic polymer.
7. The method of claim 1 , wherein the compound comprises a protein or an oligo- or polypeptide which is modified in post-translational modification.
8. The method of claim 7 , wherein the oligo- or polypeptide is modified in post-translational modification by a means comprising phosphorylation, acetylation, methylation, acylation, glycosylation, GPI anchor addition, hydroxylation, sulfation, disulfide bond formation, deamidation, or nitration.
9. The method of claim 3 , wherein the macromolecule or small biological compound comprises a lipid, a glycolipid, a lipoprotein, an apolipoprotein, a cytokine, a hormone, ceramide, a glycosylceramide, a monosaccharide ceramide, glucosylceramide, galactosylceramide, a disaccharide ceramide, lactosylceramide, a sphingosine, or a sphingolipid.
10. The method of claim 1 , wherein the functional group comprises a phosphate, an acetyl, a methyl, an acyl, a glycosyl, a sulfate, a sulfonate, a thiol, an amide, a hydroxyl or a nitro.
11. The method of claim 1 , wherein the chemical, enzymatic or catalytic source of the functional group comprises an enzyme and a chemical comprising the functional group.
12. The method of claim 11 , wherein the enzyme is a kinase, a sulfotransferase, an acetyltransferase, a methylase, a carboxylase, an aminase or an amidase.
13. The method of claim 11 , wherein the enzyme is a kinase and the chemical comprising the functional group is ATP.
14. The method of claim 1 , wherein the precipitation is carried out by means of an ion.
15. The method of claim 14 , wherein the ion comprises a metal ion.
16. The method of claim 1 , further comprising providing a normalizing reagent to promote precipitation of the compound that has gained the functional group or the compound that has not gained the functional group.
17. The method of claim 1 , further comprising resolubilizing the precipitated compound then measuring the non-radioactive signaling moiety in the resolubilized preparation.
18. The method of claim 1 , further comprising blocking at least one moiety on the compound that interferes with
the compound gaining the functional group in step (b) or
separating the compound in step (c).
19. The method of claim 2 , wherein the detecting or quantifying is carried out directly.
20. The method of claim 2 , wherein the detecting or quantifying is carried out indirectly.
21. The method of claim 1 , wherein the non-radioactive signaling moiety comprises a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chromogenic compound, a chelating compound, an electron dense compound, a magnetic compound, an energy transfer member or pair, an intercalating compound, an antibody, an antigen, a hapten, a receptor, a hormone, a ligand or an enzyme, or any combination thereof.
22. A method for isolating a compound which has lost at least one functional group, the method comprising
(a) providing:
(i) a compound comprising a functional group and being directly covalently bound to at least one labeled non-radioactive signaling moiety;
(ii) a chemical, enzymatic or catalytic source which can remove the functional group from the compound; and
(iii) means for separating the compound which has lost the functional group from the compound which has not lost the functional group;
(b) forming a mixture comprising the labeled compound and the chemical, enzymatic or catalytic source and incubating the mixture under conditions suitable for the compound to lose the functional group; and
(c) separating and thereby isolating any compound which has lost the functional group from any compound which retains the functional group by precipitating the compound which retains the functional group from the mixture; and
(d) collecting unprecipitated material.
23. The method of claim 22 , further comprising detecting or quantifying the nonradioactive signaling moiety in said compound in solution, to detect or quantify said compound which has lost the functional group.