Methods, compositions and kits are disclosed for determining one or more target polypeptides in a sample where the target polypeptides have undergone a post-translational modification. A mixture comprising the sample and a first reagent comprising a cleavage-inducing moiety and a first binding agent 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 post-translational modification 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 the post-translational modification of the target polypeptide. The interaction between the first binding agent 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.
本发明揭示了一种用于确定样品中经历了翻译后修饰的一个或多个目标
多肽的方法、组合物和试剂盒。将包含样品和第一试剂的混合物暴露在相应的结合物质发生结合的条件下,其中第一试剂包括一个裂解诱导基团和一个与目标
多肽上的结合位点结合的第一结合剂。该结合位点是涉及目标
多肽的翻译后修饰活性的结果。该方法可用于确定目标
多肽本身。在另一实施例中,目标
多肽的存在和/或数量与参与目标
多肽翻译后修饰的酶等试剂的存在、数量和/或活性相关。第一结合剂和结合位点之间的相互作用将裂解诱导基团带到可裂解基团的近距离位置,后者与
多肽相关,并且只有在接近裂解诱导基团时才易于裂解。通过这种方式,每个
多肽的电泳标签可以被释放。释放的电泳标签被分离,并根据相应的电泳标签确定目标
多肽的存在和/或数量。