Kinase/phosphatase substrate analysis and compositions using spectrally encoded microbeads
AssigneesCZ Biohub SF, LLC, The Board of Trustees of the Leland Stanford Junior University, University of Copenhagen
InventorsPolly M. Fordyce, Jamin B. Hein
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Abstract
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.
Claims23
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.
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.
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.
(canceled)
Dependencies are inferred from the claim text.