Homogeneous luminescent quantitation of cellular guanosine and adenosine triphosphates (GTP and ATP) using QT-Luc assay.

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-Luc, enabling high-throughput compatible dual monitoring of cellular GTP and ATP in a homogenous fashion. Collectively, GTP-QRET and QT-Luc 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.