CROSS-LINKING ATP ANALOGS
An adenosine 5′-triphosphate analogs modified at the gamma-phosphate with a reactive reagent. A method of forming the analog by activating a 4-amino benzoic acid, incubating the activated acid to obtain an amine, and coupling the amine with ATP in the presence of water soluble EDCI. A method of detecting the efficacy of a therapeutic by adding a gamma-phosphate modified ATP analog to a protein substrate, reacting the target proteins with the ATP analog, and analyzing the resulting cross-linked product, wherein the amount of product present correlates to the efficacy of the therapeutic is also provided.
1 . An analog comprising adenosine 5′-triphosphate analogs modified at the gamma-phosphate with a reactive reagent.
2 . The analog according to claim 1 , wherein said adenosine 5′-triphosphate analog is selected from the group consisting essentially of ATP-arylazide, ATP-benzophenone, ATP-diazirine, ATP-azide, ATP-alkyne, bi-functional crosslinking ATP analogs, and ATP-iodoacetamide.
3 . The analog according to claim 1 for use in characterizing phosphorylated proteins.
4 . The analog according to claim 1 for use in understanding protein-protein interactions.
5 . A method of forming the analog according to claim 1 comprising the steps of:
activating a 4-amino benzoic acid;
incubating the activated acid to obtain an amine; and
coupling the amine with ATP in the presence of water soluble EDCI.
6 . A kit for detecting characterizing a target protein in a sample, the kit comprising:
a gamma-phosphate modified ATP analog for selectively tagging a protein at phosphorylated amino acids with a cross-linking group.
7 . A method of using the kit according to claim 6 to characterize the target protein comprising the steps of:
adding a gamma-phosphate modified ATP analog to a protein substrate;
reacting the target proteins with the ATP analog; and
analyzing the resulting cross-linked product.
8 . The method according to claim 7 , wherein said analyzing step is performed using a method selected from the group consisting essentially of gel electrophoresis, Western blotting, SDS-PAGE, chromatography, and mass spectrometric analysis.
9 . The method according to claim 7 , further including the step of detecting abnormally phosphorylated proteins.
10 . The method according to claim 9 , further including correlating the abnormally phosphorylated protein with a specific disease state.
11 . The method according to claim 10 , further including the step of identifying potential therapeutics for use in preventing the abnormal phosphorylation and the specific disease state.
12 . A method of detecting the efficacy of a therapeutic comprising the steps of:
adding a gamma-phosphate modified ATP analog to a protein substrate;
reacting the target proteins with the ATP analog; and
analyzing the resulting cross-linked product, wherein the amount of product present correlates to the efficacy of the therapeutic.
13 . A screen identifying therapeutics, the screen comprising the kit according to claim 6 .
14 . Use of the ATP-analog of claim 1 for identification of phosphorylation-associated biomolecules.
15 . The use according to claim 14 , wherein said identification comprises the steps of:
adding a gamma-phosphate modified ATP analog to a protein substrate;
reacting the target proteins with the ATP analog;
analyzing the resulting cross-linked product; and
identifying phosphorylation-associated biomolecules based on results from said analyzing step.