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

• Published in: Analytical and bioanalytical chemistry 2023-11, Vol.415 (27), p.6689-6700
• Main Authors: Kopra, Kari, Mahran, Randa, Yli-Hollo, Titta, Tabata, Sho, Vuorinen, Emmiliisa, Fujii, Yuki, Vuorinen, Iida, Ogawa-Iio, Aki, Hirayama, Akiyoshi, Soga, Tomoyoshi, Sasaki, Atsuo T., Härmä, Harri
• Format: Article
• Language: English
• Subjects: Adenosine triphosphate; Analytical Chemistry; ATP; Biochemistry; Capillary electrophoresis; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Drug development; Electrophoresis; Energy metabolism; Energy transfer; Food Science; Guanosine triphosphate; Guanosines; Instrumentation; Laboratory Medicine; Liquid chromatography; Malignancy; Mass spectrometry; Mass spectroscopy; Metabolites; Monitoring; Monitoring/Environmental Analysis; New technology; Nucleic acids; Quantitative analysis; Research Paper
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-023-04944-9

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