Analyzing phosphorylated proteins
First Claim
1. A method for determining the presence or absence of one or more target polypeptides in a sample suspected of containing the target polypeptides, the method comprising the steps of:
- a)—
mixing the sample with a class-specific reagent and one or more electrophoretic probes, wherein the class-specific reagent comprises (i) a cleavage-inducing moiety having an effective proximity and (ii) an immobilized metal affinity chromatography (IMAC) resin that binds a phosphate group on the one or more target polypeptides, and wherein the one or more electrophoretic probes each comprises a binding moiety that specifically binds one of the target polypeptide(s) and one or more electrophoretic tags each attached thereto by a cleavable linkage, under conditions that permit the class-specific reagent and the one or more electrophoretic probes to bind to the one or more target polypeptides;
b)—
cleaving the cleavable linkage(s) of any of the one or more electrophoretic probes that are within the effective proximity of the cleavage-inducing moiety so that one or more electrophoretic tags are released; and
c)—
determining the presence or absence of the one or more target polypeptides based on the presence or absence of the released electrophoretic tag(s).
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Accused Products
Abstract
Methods, compositions and kits are disclosed for determining one or more target polypeptides in a sample where the target polypeptides have undergone phosphorylation. A mixture comprising the sample and a first reagent comprising a cleavage-inducing moiety and an IMAC resin for a binding site on a target polypeptide is subjected to conditions under which binding of respective binding moieties occurs. The binding site is the result of phosphorylation activity involving the target polypeptide. The method may be employed to determine the target polypeptide itself. In another embodiment the presence and/or amount of the target polypeptide is related to the presence and/or amount and/or activity of an agent such as an enzyme involved in phosphorylation of the target polypeptide. The interaction between the IMAC resin and the binding site brings the cleavage-inducing moiety into close proximity to a cleavable moiety, which is associated with the polypeptide and is susceptible to cleavage only when in proximity to the cleavage-inducing moiety. In this way, an electrophoretic tag for each of the polypeptides may be released. Released electrophoretic tags are separated and the presence and/or amount of the target polypeptides are determined based on the corresponding electrophoretic tags.
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Citations
22 Claims
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1. A method for determining the presence or absence of one or more target polypeptides in a sample suspected of containing the target polypeptides, the method comprising the steps of:
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a)—
mixing the sample with a class-specific reagent and one or more electrophoretic probes, wherein the class-specific reagent comprises (i) a cleavage-inducing moiety having an effective proximity and (ii) an immobilized metal affinity chromatography (IMAC) resin that binds a phosphate group on the one or more target polypeptides, and wherein the one or more electrophoretic probes each comprises a binding moiety that specifically binds one of the target polypeptide(s) and one or more electrophoretic tags each attached thereto by a cleavable linkage, under conditions that permit the class-specific reagent and the one or more electrophoretic probes to bind to the one or more target polypeptides;b)—
cleaving the cleavable linkage(s) of any of the one or more electrophoretic probes that are within the effective proximity of the cleavage-inducing moiety so that one or more electrophoretic tags are released; andc)—
determining the presence or absence of the one or more target polypeptides based on the presence or absence of the released electrophoretic tag(s). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 20, 21, 22)
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14. A kit for detecting the presence or absence of a plurality of target polypeptides in a sample, the kit comprising:
- a class-specific reagent comprising a cleavage-inducing moiety and an IMAC resin specific for a phosphate group on the one or more target polypeptides; and
one or more electrophoretic probes each comprising a binding moiety specific for a target polypeptide and one of more electrophoretic tags attached by a cleavable linkage. - View Dependent Claims (15, 16, 17, 18, 19)
- a class-specific reagent comprising a cleavage-inducing moiety and an IMAC resin specific for a phosphate group on the one or more target polypeptides; and
Specification