Method for identifying activation of transferases
Inventors
Rodriguez Cutillas, Pedro • Vanhaesebroeck, Bart • Beltran, Luisa Marie
Assignees
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Publication Number
US-9747412-B2
Publication Date
2017-08-29
Expiration Date
Abstract
The present invention provides a method for identifying differential activation of a bisubstrate protein modifying enzyme between samples, comprising: (i) incubating a first sample with x different concentrations of the non-protein substrate of said enzyme, wherein x is 2 or greater than 2;(ii) quantifying modification of a polypeptide in said sample at each of the x different concentrations of the non-protein substrate;(iii) determining the affinity of said enzyme for said non-protein substrate;(iv) repeating steps (i) to (iii) for a second or subsequent sample; and(v) comparing the affinity of said enzyme for said non-protein substrate between said samples; wherein a difference in affinity of said enzyme for said non-protein substrate between samples is indicative of differential activation of said enzyme between samples. The present invention also provides a method for identifying an in vivo substrate of a bisubstrate protein modifying enzyme.
Core Innovation
The invention describes methods for assessing differential activation between samples of a protein transferase having a non-protein donor substrate and a protein acceptor substrate. A first sample is exposed to x different concentrations of the non-protein donor substrate, modification of a polypeptide is quantified at each concentration, and the affinity of the protein transferase for the non-protein donor substrate is determined.
The affinities are compared between the first sample and one or more additional samples to indicate differential activation. The approach further identifies an in vivo substrate by exposing a protein transferase to x different concentrations of a first substrate while leaving the concentration of a second substrate constant, where one substrate is the non-protein donor substrate and the other is a mixture of polypeptides.
Modification of polypeptides in the mixture is quantified at each concentration, and the affinity of the protein transferase for the first substrate is determined. A high affinity of the protein transferase for the first substrate is indicative of the polypeptide being an in vivo substrate. The described outcomes include identification of many Akt1-dependent phosphosites, ranking by affinity constants (Ac), and detecting stimulus- and inhibitor-modulated kinase activities.
Claims Coverage
The partial content provides two independent claims covering differential activation between samples and in vivo substrate identification. Across these claims, the inventive features include affinity determination from modification quantification across multiple substrate concentrations and using affinity differences or high affinity as indicators of differential activation or in vivo substrate status.
Affinity-based differential activation measurement using multiple donor concentrations
Exposing a first sample to x different concentrations of the non-protein donor substrate of a protein transferase, quantifying modification of a polypeptide at each concentration, determining the affinity of the protein transferase for the non-protein donor substrate, repeating for additional samples, and comparing the affinity between samples, wherein a difference in affinity is indicative of differential activation.
In vivo substrate identification using high affinity under constant acceptor concentration
Exposing the protein transferase to x different concentrations of a first substrate while leaving the concentration of a second substrate constant, where one substrate is the non-protein donor substrate and the other is a mixture of polypeptides; quantifying modification of a polypeptide in the mixture at each first-substrate concentration; determining the affinity of the protein transferase for the first substrate; wherein a high affinity for the first substrate is indicative of the polypeptide being an in vivo substrate.
Across the independent claims, the core claim coverage centers on quantifying polypeptide modifications while varying substrate concentrations to determine affinity and using affinity comparison or high affinity as indicators for differential activation or in vivo substrate identification.
Stated Advantages
Enables detection of differential activation between samples of a protein transferase via affinity differences.
Enables identification of in vivo substrates by using high affinity as an indicator.
Improves substrate confidence through coupling GKAP with quantitative phosphoproteomics and subsequent analyses.
Detects stimulus- and inhibitor-modulated kinase activities in leukemia and epithelial cell lines.
Ranks substrate-related findings by affinity constants (Ac) in the described profiling outcomes.
Documented Applications
Global kinase activity profiling (GKAP) coupling in vitro kinase reactions in cell lysates to quantitative phosphoproteomics using TIQUAS/PESCAL and motif analysis.
Identification of Akt1-dependent phosphosites and ranking by affinity constants (Ac).
Profiling stimulus- and inhibitor-modulated kinase activities in leukemia cell lines (P31/Fuj, Kasumi-1).
Profiling stimulus- and inhibitor-modulated kinase activities in an epithelial cell line (MCF10A).
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