KINASE/PHOSPHATASE SUBSTRATE ANALYSIS AND COMPOSITIONS USING SPECTRALLY ENCODED MICROBEADS
The disclosure provides methods and compositions for evaluating phosphatase and kinase activity using spectrally encoded microbeads comprising substrate peptides in which there is a 1:1 linkage between sequences of the substrate peptide and the embedded spectral code.
1 . A method of assaying peptide dephosphorylation comprising providing a population comprising microbeads having distinguishable spectral codes, wherein each microbead comprises a substrate peptide attached thereto, wherein each substrate peptide comprises a phosphorylation site that contains a phosphorylated amino acid residue, and wherein each microbead having the same spectral code comprises a substrate peptide having a phosphorylation site of the same sequence and microbeads having different spectral codes comprise substrate peptides comprising phosphorylation sites of different sequences;
contacting the population of microbeads with a phosphatase;
detecting phosphorylated residues of substrate peptides on individual beads following removal of the phosphatase;
determining the spectral signature of individual microbeads in the population, thereby determining the identity of the substrate peptide linked to each bead.
2 . The method of claim 1 , wherein the distinguishable spectral codes are lanthanide spectral codes.
3 . The method of claim 2 , wherein the step of detecting phosphorylated residues comprises contacting microbeads with a labeled reagent that binds to phosphates present on phosphorylated substrate peptides.
4 . The method of claim 1 , wherein the contacting step comprises detecting a pooled population of microbeads with the phosphatase.
5 . The method of claim 4 , wherein the detecting step comprises distributing the pooled population into an array configuration.
6 . The method of claim 1 , wherein the lanthanide spectral codes are detecting by deep UV imaging.
7 . The method of claim 1 , wherein the labeled reagent that binds to phosphates comprises a fluorescent label.
8 . The method of claim 1 , wherein the labeled reagent is a biotin-linked titanium dioxide nanoparticle.
9 . The method of claim 1 , wherein phosphates are detected by image analysis.
10 . The method of claim 1 , wherein the phosphorylated substrate peptides are synthesized on the microbeads.
11 . A method of assaying substrate peptide phosphorylation comprising providing a population comprising microbeads having distinguishable spectral codes, wherein each microbead comprises a substrate peptide comprising a phosphorylation site attached thereto and wherein each microbead having the same spectral code comprises a substrate peptide comprising the same phosphorylation site sequence and microbeads having different spectral codes comprise substrate peptides comprising phosphorylation sites that differ in sequence;
contacting the population of microbeads with a kinase;
detecting phosphorylated residues of substrate peptides on individual microbeads;
determining the spectral signature of individual microbeads in the population, thereby determining the identity of the substrate peptide linked to individual beads.
12 . The method of claim 11 , wherein the distinguishable spectral codes are lanthanide spectral codes.
13 . The method of claim 11 , wherein the step of detecting phosphate residues comprises contacting microbeads with a labeled reagent that binds to phosphates present on phosphorylated substrate peptides.
14 . The method of claim 11 , wherein the contacting step comprises contacting a pooled population of microbeads with the kinase.
15 . The method of claim 14 , wherein the detecting step comprises distributing the pooled population into an array configuration.
16 . The method of claim 11 , wherein the lanthanide spectral codes are detecting by deep UV imaging.
17 . The method of claim 11 , wherein the labeled reagent that binds to phosphates comprises a fluorescent label.
18 . The method of claim 1 , wherein the labeled reagent is a biotin-linked titanium dioxide nanoparticle.
19 . The method of claim 1 , wherein phosphates are detected by image analysis.
20 . The method of claim 1 wherein the phosphorylated substrate peptides are synthesized on the microbeads.
21 . A population of lanthanide-encoded microbeads comprising microbeads having distinguishable lanthanide spectral codes, wherein each microbead comprises a substrate peptide comprising a phosphorylation site attached thereto and wherein each microbead having the same spectral code comprises a substrate peptide comprising the same phosphorylation site sequence and microbeads having different spectral codes comprise substrate peptides comprising phosphorylation sites that differ in sequence; or
a population of lanthanide-encoded microbeads comprising microbeads having distinguishable lanthanide spectral codes, wherein each microbead comprises a phosphorylated substrate peptide comprising a phosphorylation site that contains a phosphorylated amino acid residue, and wherein each microbead having the same spectral code comprises a substrate peptide comprising the same phosphorylation site sequence and microbeads having different spectral codes comprise substrate peptides comprising phosphorylation sites that differ in sequence.
22 . (canceled)
23 . The population of claim 21 , wherein the phosphorylated substrate peptides are synthesized on the microbeads.