Methods, Particles, and Kits for Determining Activity of a Kinase

Abstract

Methods, particles and kits for determining kinase activity within a sample are provided. An embodiment of a method includes exposing a fluorescent particle to an assay, wherein the fluorescent particle includes a support substrate having one or more fluorescent materials and a peptide substrate coupled to the support substrate via a functional group of the support substrate. The method further includes phosphorylating the peptide substrate during exposure of the fluorescent particle to the assay and processing the fluorescent particle such that the peptide substrate is dephosphorylated and a polarized double bond is generated at a dephosphorylated site. In addition, the method includes coupling a fluorescent reporter having a nucleophilic terminal group to the fluorescent particle via the polarized double bond.

1 . A method, comprising:
 exposing a fluorescent particle to an assay, wherein the fluorescent particle comprises:
 a support substrate comprising one or more fluorescent materials; and 
 a peptide substrate coupled to the support substrate via a functional group of the support substrate; 
   phosphorylating the peptide substrate during the step of exposing the fluorescent particle to the assay;   processing the fluorescent particle such that the peptide substrate is dephosphorylated and a polarized double bond is generated at a dephosphorylated site; and   coupling a fluorescent reporter comprising a nucleophilic terminal group to the fluorescent particle via the polarized double bond, wherein the fluorescent reporter is configured to emit fluorescence within a different wavelength range than the one or more fluorescent materials of the support substrate.   
     
     
         2 . The method of  claim 1 , wherein the fluorescent reporter comprises:
 a fluorescent compound; and   one or more spacer compounds interposed between the nucleophilic terminal group and the fluorescent compound.   
     
     
         3 . The method of  claim 2 , wherein the one or more spacer compounds collectively comprise between approximately 1 atom and approximately 25 atoms. 
     
     
         4 . The method of  claim 1 , wherein the fluorescent reporter is configured to emit fluorescence at a wavelength greater than approximately 500 nm. 
     
     
         5 . The method of  claim 1 , wherein the step of phosphorylating the peptide substrate comprises exposing the fluorescent particle to an ionic liquid heated via microwaves. 
     
     
         6 . The method of  claim 1 , wherein the step of processing the fluorescent particle comprises exposing the fluorescent particle to an ionic liquid. 
     
     
         7 . The method of  claim 6 , wherein the step of processing the fluorescent particle further comprises microwave heating the fluorescent particle and the ionic liquid. 
     
     
         8 . The method of  claim 1 , wherein the step of coupling the fluorescent reporter comprises exposing the fluorescent particle to an ionic liquid. 
     
     
         9 . The method of  claim 8 , wherein the step of coupling the fluorescent reporter further comprises microwave heating the fluorescent particle and the ionic liquid. 
     
     
         10 . A method, comprising:
 exposing a pooled population of different subsets of fluorescent particles to a sample, wherein at least some of the fluorescent particles comprise:
 a support substrate comprising one or more fluorescent materials configured to emit fluorescence in a first wavelength range, and wherein at least some of the different subsets of fluorescent particles respectively comprise a different configuration of the one or more fluorescent materials; and 
 a peptide substrate coupled to the support substrate via a functional group of the support substrate, and wherein at least some of the different subsets of fluorescent particles respectively comprise a different peptide substrate; 
   exposing the sample and the pooled population to a phosphorylation process configured to add phosphate groups to accepting residues of the peptide substrates;   processing a plurality of the fluorescent particles subsequent to exposing the sample and the pooled population to the phosphorylation process such that if any phosphorylated peptide substrates exist among the plurality of fluorescent particles, the phosphorylated peptide substrates are dephosphorylated and polarized double bonds are generated at dephosphorylated sites of the peptide substrates;   further processing the plurality of the fluorescent particles such that if any polarized double bonds exist among the dephosphorylated sites of the peptide substrates, fluorescent reporters are coupled to the fluorescent particles at positions of the polarized double bonds via nucleophilic terminal groups of the fluorescent reporters, wherein the fluorescent reporters are configured to emit fluorescence in a second wavelength range distinct from the first wavelength range;   measuring fluorescence emissions of the plurality of the fluorescent particles subsequent to further processing the plurality of the fluorescent particles;   identifying subset classifications of the particles in the sample based upon measured fluorescence emissions within the first wavelength range; and   determining, based upon the existence of or lack of measured fluorescence emissions within the second wavelength range, an amount of kinase activity within the sample when the sample and the pooled population are exposed to the phosphorylation process.   
     
     
         11 . The method of  claim 10 , wherein the step of identifying subset classifications of the particles comprises determining the identity of more than approximately 100 subset classifications. 
     
     
         12 . The method of  claim 10 , wherein the step of further processing the plurality of the fluorescent particles comprises:
 coupling a first fluorescent reporter to fluorescent particles within a first subset of the plurality of fluorescent particles; and   coupling a different fluorescent reporter to fluorescent particles within a second subset of the plurality of fluorescent particles, and wherein the different fluorescent reporter is configured to emit fluorescence in a wavelength range distinct from a wavelength range the first fluorescent reporter is configured to emit.   
     
     
         13 . A kit, comprising:
 a plurality of fluorescent particles, wherein each of the plurality of fluorescent particles comprises a support substrate with one or more fluorescent materials configured to emit fluorescence in a first wavelength range; and   a plurality of kinase-specific peptide substrates respectively coupled to different subsets of the plurality of fluorescent particles via functional groups of the support substrates.   
     
     
         14 . The kit of  claim 13 , further comprising:
 a phosphorylation reagent configured to phosphorylate the kinase-specific peptide substrates; and   a beta-elimination reagent configured to dephosphorylate the kinase-specific peptide substrates and generate Michael acceptors at the dephosphorylation sites of the kinase-specific peptide substrates.   
     
     
         15 . The kit of  claim 14 , wherein at least one of the phosphorylation reagent and the beta-elimination reagent comprises an ionic liquid. 
     
     
         16 . The kit of  claim 13 , further comprising one or more fluorescent reporter reagents each having a nucleophilic terminal group and one or more fluorescent compounds configured to emit fluorescence in a wavelength range distinct from the first wavelength range. 
     
     
         17 . The kit of  claim 16 , wherein the one or more fluorescent report reagents comprise a plurality of fluorescent report reagents which are respectively configured to emit fluorescence in a different wavelength range. 
     
     
         18 . The kit of  claim 16 , wherein at least one of the one or more fluorescent reporter reagents comprises a hydrophilic fluorescent compound. 
     
     
         19 . The kit of  claim 16 , wherein at least one of the one or more fluorescent reporter reagents comprises an ionic liquid.