Molecular modification assays involving lipids
Assays for detecting molecular modifications, such as phosphorylation, dephosphorylation, and/or cleavage, among others, of lipids, lipid fragments, and/or lipid precursors. These assays may be used to detect the presence and/or activity of enzymes and/or other agents, such as drugs, involved in facilitating, inhibiting, and/or otherwise regulating such modifications.
1 . A method of detecting modification of a lipid or lipid precursor in a sample, comprising:
contacting the sample with a binding partner that binds specifically to a modified form or a nonmodified form of the lipid or lipid precursor, but not both, the binding partner including a metal required for specific binding to the one form; and
detecting a response indicative of the extent of binding between the binding partner and the one form.
2 . The method of claim 1 , wherein the detected response is indicative of a decrease in the nonmodified form of the lipid.
3 . The method of claim 1 , wherein the detected response is indicative of the presence of the nonmodified form of the lipid.
4 . The method of claim 1 , wherein the detected response includes a luminescence polarization response.
5 . The method of claim 1 , wherein the detected response includes a luminescence resonance energy transfer response.
6 . The method of claim 1 , wherein the detected response includes at least one of a change in the intensity, polarization, energy transfer, lifetime, and excitation or emission wavelength distribution of a detected luminescence.
7 . The method of claim 1 , wherein the modified and nonmodified forms are related by an enzymatic modification.
8 . The method of claim 7 , the enzymatic modification being mediated by an enzyme, further comprising adding a candidate modulator of activity of the enzyme to the sample, prior to the step of detecting a response.
9 . The method of claim 7 , wherein the enzymatic modification is cleavage.
10 . The method of claim 9 , wherein the enzymatic modification cleaves the lipid or the lipid precursor between a phosphate moiety and a luminescent moiety.
11 . The method of claim 7 , wherein the enzymatic modification is phosphorylation.
12 . The method of claim 1 , wherein the extent of binding between the binding partner and the one form is related to one or more functional groups of the one form.
13 . The method of claim 12 , wherein the functional group is ionic at physiological pH.
14 . The method of claim 12 , wherein the functional group is selected from phosphate, sulfonate, and carboxylate.
15 . The method of claim 1 , wherein the binding partner includes a cationic metal ion.
16 . The method of claim 15 , wherein the binding partner includes a gallium ion.
17 . The method of claim 15 , wherein the binding partner includes a particle or macromolecule associated with the metal ion.
18 . The method of claim 1 , wherein the method is performed with a lipid, and wherein the lipid is a phosphoinositide.
19 . The method of claim 1 , wherein the method if performed with a lipid precursor, and wherein the lipid precursor includes inositol.
20 . The method of claim 1 , wherein the step of contacting also contacts the binding partner with a luminescent reporter to which the binding partner can bind, and wherein the step of detecting includes a step of measuring competition, if any, between the one form and the luminescent reporter for binding to the binding partner.
21 . The method of claim 1 , wherein the step of contacting is performed in the presence of a detergent that increases the response relative to no detergent.
22 . A kit configured to perform the method of claim 1.