Methods for determination of protein phosphatase activity, and uses in predicting therapeutic outcomes

Abstract

One aspect of the present disclosure encompasses methods for determining a protein kinase or phosphatase activity in a biological sample, comprising: contacting in a reaction mix a first test sample and a fluorescently-labeled peptide substrate capable of being modified by a protein phosphatase or a protein kinase, contacting the reaction mix with a TiO 2 matrix, thereby partitioning fluorescently-labeled phosphorylated peptide from fluorescently-labeled dephosphorylated peptide; and determining the fluorescence of the fluorescently-labeled dephosphorylated peptide, thereby determining a protein kinase or phosphatase activity.

1 . A method for determining a protein kinase or phosphatase activity in a biological sample, comprising:
 contacting in a reaction mix a first test sample and a fluorescently-labeled peptide substrate capable of being modified by a protein phosphatase or a protein kinase, under conditions allowing kinase or phosphatase to modify the phosphorus status of the peptide;   contacting the reaction mix with a TiO 2  matrix, thereby partitioning fluorescently-labeled phosphorylated peptide from fluorescently-labeled non-phosphorylated peptide;   determining the fluorescence of the fluorescently-labeled non-phosphorylated peptide, thereby determining a protein kinase or phosphatase activity.   
     
     
         2 . The method of  claim 1 , wherein the steps further comprise:
 providing a first test sample;   admixing a fluorescently-labeled phosphorylated peptide substrate, a reaction buffer, and a first test sample to form a first reaction mix;   incubating the first reaction mix under conditions allowing a phosphatase to dephosphorylate the fluorescently-labeled phosphorylated peptide;   providing a reaction vessel, wherein the reaction vessel is coated with a TiO 2  matrix, and wherein the TiO 2  matrix is contacted with a binding buffer;   delivering the first reaction mix to the coated vessel, and incubating under conditions allowing binding of fluorescently-labeled phosphorylated peptide to the TiO 2  matrix;   transferring the first reaction mix from the coated well to a vessel containing ammonium hydroxide; and   determining the amount of fluorescence emitted by the fluorescently-labeled dephosphorylated peptide of the first reaction mix.   
     
     
         3 . The method of  claim 1 , wherein the steps further comprise:
 providing a first test sample;   admixing a fluorescently-labeled non-phosphorylated peptide substrate, a reaction buffer, and a first test sample to form a first reaction mix;   incubating the first reaction mix under conditions allowing a kinase to phosphorylate the fluorescently-labeled phosphorylated peptide;   providing a reaction vessel, wherein the reaction vessel is coated with a TiO 2  matrix, and wherein the TiO 2  matrix is contacted with a binding buffer;   delivering the first reaction mix to the coated vessel, and incubating under conditions allowing binding of fluorescently-labeled phosphorylated peptide to the TiO 2  matrix;   transferring the first reaction mix from the coated well to a vessel containing ammonium hydroxide; and   determining the amount of fluorescence emitted by the fluorescently-labeled dephosphorylated peptide of the first reaction mix.   
     
     
         4 . The assay method of  claim 1 , wherein the fluorescently labeled phosphorylated peptide has an amino acid sequence selected from SEQ ID NO.: 1 and SEQ ID NO.: 2. 
     
     
         5 . The assay method of  claim 1 , wherein the fluorescently labeled phosphorylated peptide is capable of distinguishing a first isoform of calcineurin from a second isoform. 
     
     
         6 . The assay method of  claim 1 , wherein the fluorescently labeled phosphorylated peptide is capable of being specifically dephosphorylated by the β-isoform of calcineurin and has the amino acid sequence according to SEQ ID NO.: 2. 
     
     
         7 . The assay method of  claim 1 , wherein the fluorescently labeled phosphorylated peptide has the amino acid sequence according to SEQ ID NO.: 1, is phosphorylated on the Ser-15 position, and further comprises an N-terminal fluorescein group. 
     
     
         8 . The assay method of  claim 2 , wherein the fluorescently labeled phosphorylated peptide capable of being specifically dephosphorylated by the β-isoform of calcineurin comprises a peptide having the amino acid sequence according to SEQ ID NO.: 2, wherein the S-6 position is phosphorylated, an N-terminal fluorescent TAMRA group, and a C-terminal amide group. 
     
     
         9 . The assay method of  claim 2 , further comprising comparing the fluorescence emitted by the first reaction mix with the fluorescence emitted by at least one second test sample comprising a known amount of active calcineurin, thereby determining the amount of calcineurin in the first test sample. 
     
     
         10 . The assay method of  claim 2 , further comprising comparing the fluorescence emitted by the first reaction mix with the fluorescence emitted by at least one second test sample, wherein the second test sample includes a calcineurin inhibitor. 
     
     
         11 . The assay method of  claim 2 , wherein the assay method is configured for high-throughput screening of a plurality of test samples by providing a plurality of test samples, thereby forming a plurality of reaction mixes. 
     
     
         12 . The method of  claim 2 , wherein the reaction vessel is a well of a multi-well assay plate. 
     
     
         13 . The method of  claim 2  configured for predicting the outcome of a transplant in a patient in need thereof, wherein:
 the first test sample is obtained from a patient, wherein the patient is in need of a transplant or has received a transplant; and   determining from the level of calcineurin activity in the test sample in the presence of a calcineurin inhibitor the efficacy of the calcineurin inhibitor in the patient.   
     
     
         14 . A method of determining the response of calcineurin of a human or animal patient to a calcineurin inhibitor, comprising:
 obtaining from a patient a first cell or tissue test sample and a second cell or tissue sample;   determining the level of activity of calcineurin in the first test sample;   determining the level of activity of calcineurin in the second test sample in the presence of a calcineurin inhibitor; and   comparing the levels of calcineurin activity in the first and second samples, thereby predicting a response of the patient to a calcineurin inhibitor administered thereto.   
     
     
         15 . The method of  claim 14 , wherein the prediction of the response of a patient to an administered calcineurin inhibitor further predicts the likely outcome of a transplant in the patient. 
     
     
         16 . The method of  claim 15 , wherein the transplant is a renal transplant. 
     
     
         17 . The method of  claim 14 , wherein the step of determining the level of activity of calcineurin in the test sample comprises the steps of
 contacting in a reaction mix a first test sample and a fluorescently labeled phosphorylated peptide substrate capable of being dephosphorylated by calcineurin, under conditions allowing calcineurin to dephosphorylate the fluorescently labeled phosphorylated peptide;   contacting the reaction mix with a TiO 2  matrix, thereby partitioning fluorescently labeled phosphorylated peptide from fluorescently labeled dephosphorylated peptide;   determining the fluorescence of the fluorescently labeled dephosphorylated peptide, thereby detecting calcineurin activity.   
     
     
         18 . The method of  claim 14 , wherein the steps of determining the level of activity of calcineurin in the test sample further comprise:
 providing a first test sample;   admixing a fluorescently labeled phosphorylated peptide substrate, a reaction buffer, and a first test sample to form a first reaction mix;   incubating the first reaction mix under conditions allowing calcineurin to dephosphorylate the fluorescently labeled phosphorylated peptide;   providing a reaction vessel, wherein the reaction vessel is coated with a TiO 2  matrix, and wherein the TiO 2  matrix is contacted with a binding buffer;   delivering the first reaction mix to the coated vessel, and incubating under conditions allowing binding of fluorescently labeled phosphorylated peptide to the TiO 2  matrix;   transferring the first reaction mix from the coated well to a vessel containing ammonium hydroxide; and   determining the amount of fluorescence emitted by fluorescently labeled phosphorylated peptide of the first reaction mix.   
     
     
         19 . The assay method of  claim 14 , further comprising comparing the level of fluorescence emitted by the first reaction mix with the level of fluorescence emitted by at least one second test sample comprising a known amount of active calcineurin, thereby determining the amount of calcineurin in the first test sample. 
     
     
         20 . The assay method of  claim 14 , wherein the assay method is configured for high-throughput screening of a plurality of test samples by providing a plurality of test samples, thereby forming a plurality of reaction mixes. 
     
     
         21 . The assay method of  claim 14 , wherein the fluorescently labeled phosphorylated peptide has an amino acid sequence selected from SEQ ID NO.: 1 and SEQ ID NO.: 2. 
     
     
         22 . The assay method of  claim 14 , wherein the fluorescently labeled phosphorylated peptide has the amino acid sequence according to SEQ ID NO.: 1, is phosphorylated on the Ser-15 position, and further comprises an N-terminal fluorescein group. 
     
     
         23 . The assay method of  claim 22 , wherein the fluorescently labeled phosphorylated peptide is capable of being specifically dephosphorylated by the β-isoform of calcineurin and comprises a peptide having the amino acid sequence according to SEQ ID NO.: 2, wherein the S-6 position is phosphorylated, an N-terminal fluorescent TAMRA group, and a C-terminal amide group 20. 
     
     
         24 . A kit for determining the level of a phosphatase or a kinase activity in a test sample, comprising a container enclosing a fluorescently labeled peptide substrate; and instructions for the use of the peptide in determining the phosphatase or a kinase activity of a test sample. 
     
     
         25 . The kit of  claim 24 , further comprising a reaction vessel coated with a titanium dioxide matrix. 
     
     
         26 . The kit of  claim 24 , wherein the peptide substrate is phosphorylated, and the instructions direct the use of the kit to determine a phosphatase activity. 
     
     
         27 . The kit of  claim 24 , wherein the peptide substrate is non-phosphorylated, and the instructions direct the use of the kit to determine a kinase activity. 
     
     
         28 . The kit of  claim 24 , further comprising a reaction vessel coated with a titanium dioxide matrix. 
     
     
         29 . The kit of  claim 24 , wherein the coated reaction vessel is a well in a multi-well plate 
     
     
         30 . The kit of  claim 24 , wherein the fluorescently labeled peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs.: 1 and 2. 
     
     
         31 . The assay method of  claim 24 , wherein the fluorescently labeled phosphorylated peptide has the amino acid sequence according to SEQ ID NO.: 1, is phosphorylated on the Ser-15 position, and further comprises an N-terminal fluorescein group. 
     
     
         32 . The assay method of  claim 24 , wherein the fluorescently labeled phosphorylated peptide is capable of being specifically dephosphorylated by the β-isoform of calcineurin and comprises a peptide having the amino acid sequence according to SEQ ID NO.: 2, wherein the S-6 position is phosphorylated, an N-terminal fluorescent TAMRA group, and a C-terminal amide group.20. 
     
     
         33 . The kit of  claim 24 , wherein the kit further comprises at least one of the group consisting of a reaction buffer; a binding buffer; ammonium hydroxide solution; a white wall reaction vessel, wherein the white wall reaction vessel is optionally a well of a multi-well plate; and at least one calcineurin activity standard solution.