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 detecting or quantifying an activity in a sample that causes a compound to gain a functional group, the method comprising the steps of:
(a) providing:
(i) a compound which can gain a functional group, the compound comprising at least one non-radioactive signaling moiety;
(ii) a chemical source of the functional group which can be gained by the compound; and
(iii) a sample;
(b) forming a mixture comprising the compound, the source of the functional group and the sample, and incubating the mixture under conditions suitable for the compound to gain the functional group if the activity is present in the sample; and
(c) capturing any compound that has gained the functional group and separating said compound which has gained the functional group from any compound which has not gained the functional group; and
(d) detecting or quantifying the non-radioactive signaling moiety of any captured separated compounds having the functional group, thereby detecting or quantifying the activity in the sample.
2 . The method of claim 1 , wherein the capturing and separating step comprises capturing any compound that has gained the functional group on a matrix.
3 . The method of claim 2 , wherein the matrix comprises a metal ion.
4 . The method of claim 3 , wherein the metal ion is Fe +3 , Ga +2 , Zn +3 , Zn +2 , or Mn +2 .
5 . The method of claim 1 , wherein the compound comprises a macromolecule or a small biological compound.
6 . The method of claim 5 , 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.
7 . The method of claim 5 , wherein the macromolecule or small l biological compound comprises an oligomer or a polymer.
8 . 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.
9 . The method of claim 1 , wherein the compound comprises a protein or an oligo- or polypeptide which is modified in post-translational modification.
10 . The method of claim 9 , 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.
11 . The method of claim 5 , 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.
12 . The method of claim 1 , wherein the compound comprises a non-biological compound.
13 . The method of claim 1 , wherein the functional group is a phosphate, an acetyl, a methyl, an acyl, a glycosyl, a sulfate, a sulfonate, a thiol, an amine, an amide, a hydroxyl or a nitro group.
14 . The method of claim 13 , wherein the functional group is a phosphate and the chemical source of the functional group is ATP.
15 . 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.
16 . The method of claim 15 , wherein
the capturing and separating step comprises capturing any compound that has gained the functional group on a matrix; and
the functional group comprises a phosphate, an acetyl, a methyl, an acyl, a glycosyl, a sulfate, a sulfonate, a thiol, an amine, an amide, a hydroxyl or a nitro group.
17 . A method for detecting or quantifying an activity in sample which removes a functional group from a compound, the method comprising the steps of:
(a) providing:
(i) a compound comprising a functional group and at least one non-radioactive signaling moiety; and
(ii) a sample;
(b) forming a mixture comprising the compound and the sample and incubating the mixture under conditions suitable for the compound to lose the functional group if the activity is present in the sample; and
(c) capturing and separating any compound that has the functional group following the incubation from any compound which has lost the functional group; and
(d) detecting or quantifying the non-radioactive signaling moiety of any captured separated compounds having the functional group, thereby detecting or quantifying the activity in the sample.
18 . The method of claim 17 , wherein the capturing and separating step comprises capturing any compound that has gained the functional group on a matrix.
19 . The method of claim 18 , wherein the matrix comprises a metal ion.
20 . The method of claim 93 , wherein the metal ion is Fe +3 , Ga +2 , Zn +3 , Zn +2 , or Mn +2 .
21 . The method of claim 17 , wherein the functional group is a phosphate, an acetyl, a methyl, an acyl, a glycosyl, a sulfate, a sulfonate, a thiol, an amine, an amide, a hydroxyl or a nitro group.
22 . The method of claim 17 , 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.
23 . The method of claim 22 , wherein
the capturing and separating step comprises capturing any compound that has gained the functional group on a matrix; and
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,