鸟苷三磷酸（GTP）和腺苷三磷酸（ATP）是必需的核酸构建单元，同时也是细胞反应的能量分子。细胞内GTP浓度与ATP独立波动，并在许多肿瘤中显著升高，从而促进了恶性病变的发生。定量测量ATP和GTP的浓度变化对于阐明细胞功能调控的机制变得越来越重要。液相色谱-质谱联用（LC-MS）和毛细管电泳-质谱联用（CE-MS）是广泛应用于生物代谢物鉴定和定量的强大方法。然而，这些方法在专业设备和专业知识、通量低和高成本等方面存在限制。在本文中，我们介绍了一种新颖的GTP浓度监测定量方法——GTP淬灭共振能量转移（QRET），适用于同质细胞提取物。通过CE-MS分析以及对细胞GTP水平进行药理学控制，结果显示，GTP-QRET具有较高的动态范围和准确性。此外，我们将GTP-QRET与基于荧光素酶的ATP检测相结合，形成一种新技术，称为QT-LucGTP&ATP，能够以同质方式进行高通量的细胞GTP和ATP双重监测。总体而言，GTP-QRET和QT-LucGTP&ATP提供了一种独特的、高通量的探索细胞能量代谢的机会，成为开发新型治疗方法的强大平台，并可在各个学科领域中发挥其作用。© 2023. 作者们。

Guanosine triphosphate (GTP) and adenosine triphosphate (ATP) are essential nucleic acid building blocks and serve as energy molecules for a wide range of cellular reactions. Cellular GTP concentration fluctuates independently of ATP and is significantly elevated in numerous cancers, contributing to malignancy. Quantitative measurement of ATP and GTP has become increasingly important to elucidate how concentration changes regulate cell function. Liquid chromatography-coupled mass spectrometry (LC-MS) and capillary electrophoresis-coupled MS (CE-MS) are powerful methods widely used for the identification and quantification of biological metabolites. However, these methods have limitations related to specialized instrumentation and expertise, low throughput, and high costs. Here, we introduce a novel quantitative method for GTP concentration monitoring (GTP-quenching resonance energy transfer (QRET)) in homogenous cellular extracts. CE-MS analysis along with pharmacological control of cellular GTP levels shows that GTP-QRET possesses high dynamic range and accuracy. Furthermore, we combined GTP-QRET with luciferase-based ATP detection, leading to a new technology, termed QT-LucGTP&ATP, enabling high-throughput compatible dual monitoring of cellular GTP and ATP in a homogenous fashion. Collectively, GTP-QRET and QT-LucGTP&ATP offer a unique, high-throughput opportunity to explore cellular energy metabolism, serving as a powerful platform for the development of novel therapeutics and extending its usability across a range of disciplines.© 2023. The Author(s).