Department of Chemical Engineering, Soongsil University, Seoul, South Korea.
Nat Protoc. 2013 Sep;8(9):1730-42. doi: 10.1038/nprot.2013.104. Epub 2013 Aug 15.
Identifying the peptidases that inactivate bioactive peptides (e.g., peptide hormones and neuropeptides) in mammals is an important unmet challenge. This protocol describes a recent approach that uses liquid chromatography-mass spectrometry (LC-MS) peptidomics to identify endogenous cleavage sites of a bioactive peptide; it also addresses the subsequent biochemical purification of a candidate peptidase on the basis of these cleavage sites and the validation of the candidate peptidase's role in the physiological regulation of the bioactive peptide by examining a peptidase-knockout mouse. We highlight the successful application of this protocol in the discovery that insulin-degrading enzyme (IDE) regulates physiological calcitonin gene-related peptide (CGRP) levels, and we detail the key stages and steps in this approach. This protocol requires 7 d of work; however, the total time for this protocol is highly variable because of its dependence on the availability of biological reagents such as purified enzymes and knockout mice. The protocol is valuable because it expedites the characterization of mammalian peptidases, such as IDE, which in certain instances can be used to develop novel therapeutics.
鉴定在哺乳动物中使生物活性肽（例如肽激素和神经肽）失活的肽酶是一个重要的未满足的挑战。本协议描述了一种最近的方法，该方法使用液相色谱-质谱（LC-MS）肽组学来鉴定生物活性肽的内源性切割位点；它还解决了随后根据这些切割位点对候选肽酶进行生化纯化的问题，并通过检查肽酶敲除小鼠来验证候选肽酶在生物活性肽的生理调节中的作用。我们强调了该协议在发现胰岛素降解酶（IDE）调节生理降钙素基因相关肽（CGRP）水平方面的成功应用，并详细介绍了该方法的关键阶段和步骤。该协议需要 7 天的工作时间；然而，由于其对生物试剂（如纯化酶和敲除小鼠）的可用性的依赖，该协议的总时间变化很大。该协议很有价值，因为它可以加速鉴定哺乳动物肽酶（如 IDE），在某些情况下，这些肽酶可用于开发新的治疗方法。
