Cologna Camila T, Gilles Nicolas, Echterbille Julien, Degueldre Michel, Servent Denis, de Pauw Edwin, Quinton Loïc
Laboratory of Mass Spectrometry, Department of Chemistry, University of Liège, 4000, Liège, Belgium.
Anal Bioanal Chem. 2015 Jul;407(18):5299-307. doi: 10.1007/s00216-015-8692-4. Epub 2015 May 3.
G-protein-coupled receptors (GPCRs) constitute the largest family of transmembrane proteins. Although implicated in almost all physiological processes in the human body, most of them remain unexploited, mostly because of the lack of specific ligands. The objective of this work is to develop a new mass-spectrometry-based technique capable of identifying new peptide ligands for GPCRs. The strategy is based on the incubation of cellular membranes overexpressing GPCRs with a mixture of peptides that contains potential ligands. Peptide ligands bind to the receptors, whereas other peptides remain in the binding buffer. Bound peptides are eluted from membranes and directly detected, identified, and characterized by MALDI TOF-TOF. The results reveal the efficacy of the procedure for selecting a specific ligand of GPCRs in both simple and complex mixtures of peptides. This new approach may offer direct purification, identification, and characterization of the new ligand in a single workflow. The proposed method is labeling-free and, unlike radio-binding and other techniques, it does not require a previously known labeled ligand of the studied GPCR. All these properties greatly reduce the experimental constraints. Moreover, because it is not based on the principle of a competitive specific binding, this technique constitutes a new tool to discover new ligands not only for known GPCRs, but also for orphan GPCRs.
G蛋白偶联受体（GPCRs）构成了最大的跨膜蛋白家族。尽管它们几乎涉及人体的所有生理过程，但其中大多数仍未被开发利用，主要原因是缺乏特异性配体。这项工作的目标是开发一种基于质谱的新技术，能够识别GPCRs的新肽配体。该策略基于将过表达GPCRs的细胞膜与含有潜在配体的肽混合物一起孵育。肽配体与受体结合，而其他肽则留在结合缓冲液中。结合的肽从膜上洗脱下来，然后通过基质辅助激光解吸电离飞行时间串联质谱（MALDI TOF-TOF）直接进行检测、鉴定和表征。结果揭示了该程序在简单和复杂肽混合物中选择GPCRs特异性配体的有效性。这种新方法可以在单个工作流程中直接对新配体进行纯化、鉴定和表征。所提出的方法无需标记，并且与放射性结合和其他技术不同，它不需要预先知道所研究GPCR的标记配体。所有这些特性都大大减少了实验限制。此外，由于它不是基于竞争性特异性结合的原理，该技术不仅构成了一种发现已知GPCRs新配体的新工具，也构成了一种发现孤儿GPCRs新配体的新工具。
