Experimental Medicine and Immunotherapeutics, University of Cambridge, Level 6, Centre for Clinical Investigation, Addenbrooke's Hospital, Cambridge, UK.
Experimental Medicine and Immunotherapeutics, University of Cambridge, Level 6, Centre for Clinical Investigation, Addenbrooke's Hospital, Cambridge, UK; Sosei Heptares, Granta Park, Cambridge, UK; Metabolic Research Laboratories, Institute of Metabolic Sciences, University of Cambridge, Addenbrooke's Hospital, Cambridge, CB2 0QQ, UK.
Peptides. 2021 Nov;145:170642. doi: 10.1016/j.peptides.2021.170642. Epub 2021 Aug 26.
Elabela/Toddler (ELA) is a novel endogenous ligand of the apelin receptor, whose signalling has emerged as a therapeutic target, for example, in cardiovascular disease and cancer. Shorter forms of ELA-32 have been predicted, including ELA-21 and ELA-11, but metabolism and stability of ELA-32 in humans is poorly understood. We, therefore, developed an LC-MS/MS assay to identify ELA-32 metabolites in human plasma and tissues.
Human kidney homogenates or plasma were incubated at 37 °C with ELA-32 and aliquots withdrawn over 2-4 h into guanidine hydrochloride. Proteins were precipitated and supernatant solid-phase extracted. Peptides were extracted from coronary artery, brain and kidney by immunoprecipitation or solid-phase extraction following acidification. All samples were reduced and alkylated before analysis on an Orbitrap mass spectrometer in high and nano flow mode.
The half-life of ELA-32 in plasma and kidney were 47.2 ± 5.7 min and 44.2 ± 3 s, respectively. Using PEAKS Studio and manual data analysis, the most important fragments of ELA-32 with potential biological activity identified were ELA-11, ELA-16, ELA-19 and ELA-20. The corresponding fragments resulting from the loss of C-terminal amino acids were also identified. Endogenous levels of these peptides could not be measured, as ELA peptides are prone to oxidation and poor chromatographic peaks.
The relatively long ELA plasma half-life observed and identification of a potentially more stable fragment, ELA-16, may suggest that ELA could be a better tool compound and novel template for the development of new drugs acting at the apelin receptor.
Elabela/Toddler（ELA）是孤啡肽受体的一种新型内源性配体，其信号传导已成为治疗靶点，例如在心血管疾病和癌症中。已经预测出 ELA-32 的较短形式，包括 ELA-21 和 ELA-11，但人类 ELA-32 的代谢和稳定性知之甚少。因此，我们开发了一种 LC-MS/MS 测定法来鉴定人血浆和组织中的 ELA-32 代谢物。
将人肾匀浆或血浆在 37°C 下与 ELA-32 孵育，并在 2-4 小时内将等分试样加入盐酸胍中。沉淀蛋白质，并用固相萃取法提取上清液。在用酸处理后，通过免疫沉淀或固相萃取从冠状动脉、脑和肾中提取肽。所有样品均在高和纳流模式下的 Orbitrap 质谱仪上进行分析之前进行还原和烷基化。
ELA-32 在血浆和肾脏中的半衰期分别为 47.2±5.7 分钟和 44.2±3 秒。使用 PEAKS Studio 和手动数据分析，鉴定出具有潜在生物学活性的 ELA-32 的最重要片段为 ELA-11、ELA-16、ELA-19 和 ELA-20。还鉴定出从 C 末端氨基酸缺失产生的相应片段。由于 ELA 肽容易氧化且色谱峰较差，因此无法测量这些肽的内源性水平。
观察到的 ELA 血浆半衰期相对较长，以及鉴定出潜在更稳定的片段 ELA-16，可能表明 ELA 可能是一种更好的工具化合物和新型模板，可用于开发作用于孤啡肽受体的新药。
