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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.

Titel:	Homogeneous luminescent quantitation of cellular guanosine and adenosine triphosphates (GTP and ATP) using QT-Luc <superscript>GTP&ATP</superscript> assay.
Autor/in / Beteiligte Person:	Kopra, K ; Mahran, R ; Yli-Hollo, T ; Tabata, S ; Vuorinen, E ; Fujii, Y ; Vuorinen, I ; Ogawa-Iio, A ; Hirayama, A ; Soga, T ; Sasaki, AT ; Härmä, H
Link:	Zum Volltext
Zeitschrift:	Analytical and bioanalytical chemistry, Jg. 415 (2023-11-01), Heft 27, S. 6689-6700
Veröffentlichung:	Heidelberg : Springer-Verlag, 2002-, 2023
Medientyp:	academicJournal
ISSN:	1618-2650 (electronic)
DOI:	10.1007/s00216-023-04944-9
Schlagwort:	Guanosine Triphosphate chemistry Guanosine Triphosphate metabolism Guanosine Chromatography, Liquid Adenosine Triphosphate metabolism Adenosine
Sonstiges:	Nachgewiesen in: MEDLINE Sprachen: English Publication Type: Journal Article Language: English [Anal Bioanal Chem] 2023 Nov; Vol. 415 (27), pp. 6689-6700. <i>Date of Electronic Publication: </i>2023 Sep 16. MeSH Terms: Adenosine Triphosphate* / metabolism ; Adenosine* ; Guanosine Triphosphate / chemistry ; Guanosine Triphosphate / metabolism ; Guanosine ; Chromatography, Liquid References: Anal Chem. 2017 Apr 18;89(8):4508-4516. (PMID: 28318223) ; F1000Res. 2017 May 18;6:724. (PMID: 28663786) ; Anal Chem. 2020 Apr 7;92(7):4971-4979. (PMID: 32106676) ; J Inherit Metab Dis. 1994;17(1):138-42. (PMID: 8051925) ; Anal Chem. 2002 May 15;74(10):2233-9. (PMID: 12038746) ; Anal Bioanal Chem. 2019 Oct;411(26):6899-6911. (PMID: 31407049) ; Anal Chem. 2000 Mar 15;72(6):1236-41. (PMID: 10740865) ; J Chromatogr. 1991 Sep 18;570(1):77-88. (PMID: 1797838) ; Nat Cell Biol. 2019 Aug;21(8):1003-1014. (PMID: 31371825) ; Biochim Biophys Acta. 2008 May;1783(5):673-94. (PMID: 18302942) ; J Inherit Metab Dis. 1997 Jun;20(2):171-8. (PMID: 9211189) ; J Biol Chem. 2006 Jun 16;281(24):16768-76. (PMID: 16608839) ; Biochim Biophys Acta. 2000 Feb 21;1500(2):197-203. (PMID: 10657589) ; Anal Chim Acta. 2019 Dec 27;1092:93-101. (PMID: 31708038) ; Mol Cell. 2016 Jan 21;61(2):187-98. (PMID: 26774281) ; Nat Protoc. 2014 Aug;9(8):1944-55. (PMID: 25058642) ; Science. 2007 Apr 27;316(5824):593-7. (PMID: 17379776) ; Electrophoresis. 2020 Mar;41(5-6):360-369. (PMID: 31907937) ; Methods Mol Biol. 2022;2394:163-169. (PMID: 35094327) ; Invest Ophthalmol Vis Sci. 2006 Jan;47(1):34-42. (PMID: 16384941) ; Chem Rev. 2021 Jul 14;121(13):8095-8160. (PMID: 34105942) ; Genes Environ. 2018 Jul 30;40:13. (PMID: 30069278) ; Int J Mol Sci. 2023 May 26;24(11):. (PMID: 37298245) ; Orphanet J Rare Dis. 2007 Dec 08;2:48. (PMID: 18067674) ; J Hist Neurosci. 2005 Mar;14(1):1-10. (PMID: 15804753) ; Anal Chem. 2015 Mar 17;87(6):3527-34. (PMID: 25707436) ; Cancers (Basel). 2019 Sep 11;11(9):. (PMID: 31514446) ; Cell Rep. 2017 May 16;19(7):1313-1321. (PMID: 28514652) ; J Am Soc Mass Spectrom. 2019 Jun;30(6):987-1000. (PMID: 30847833) ; Electrophoresis. 2005 Apr;26(7-8):1424-31. (PMID: 15761917) ; Anal Bioanal Chem. 2014 Jul;406(17):4147-56. (PMID: 24760397) ; ACS Appl Mater Interfaces. 2016 Aug 17;8(32):20583-90. (PMID: 27420773) ; J Chromatogr A. 2007 Oct 19;1168(1-2):237-46; discussion 236. (PMID: 17376458) ; J Biochem. 2020 Oct 1;168(4):319-328. (PMID: 32702086) ; J Chromatogr B Analyt Technol Biomed Life Sci. 2008 Aug 1;871(1):115-24. (PMID: 18650133) ; Anal Biochem. 2006 Jan 15;348(2):243-51. (PMID: 16325757) ; Anal Biochem. 1986 Nov 15;159(1):73-81. (PMID: 3028213) ; Cancer Res. 2009 Jun 1;69(11):4918-25. (PMID: 19458066) ; Electrophoresis. 2007 Feb;28(3):373-80. (PMID: 17203505) ; Hum Mol Genet. 2002 Mar 1;11(5):547-57. (PMID: 11875049) ; Front Pharmacol. 2011 Aug 25;2:49. (PMID: 21904528) ; Arabidopsis Book. 2002;1:e0018. (PMID: 22303196) ; Elife. 2020 Apr 07;9:. (PMID: 32254022) ; J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Jan 1;1188:123044. (PMID: 34864423) ; Anal Chem. 2009 Aug 1;81(15):6165-74. (PMID: 19522513) ; Anal Biochem. 2015 Apr 15;475:22-8. (PMID: 25613493) ; Hum Mol Genet. 2002 Mar 1;11(5):559-68. (PMID: 11875050) ; Mol Cell Biochem. 1994 Nov 9;140(1):1-22. (PMID: 7877593) ; Anal Chem. 2012 Jul 17;84(14):5838-44. (PMID: 22697410) ; Nano Lett. 2009 Sep;9(9):3258-61. (PMID: 19645478) ; J Proteome Res. 2003 Sep-Oct;2(5):488-94. (PMID: 14582645) ; Keio J Med. 2016;65(1):21. (PMID: 27040886) ; ACS Chem Biol. 2008 Aug 15;3(8):499-511. (PMID: 18707057) ; Plant Physiol. 2020 Jan;182(1):63-78. (PMID: 31641078) ; Metabolomics. 2010 Mar;6(1):78-95. (PMID: 20300169) ; Medicine (Baltimore). 2001 Mar;80(2):102-12. (PMID: 11307586) ; Methods Mol Biol. 2007;358:129-37. (PMID: 17035684) Grant Information: R01 CA255331 United States CA NCI NIH HHS; R01 GM144426 United States GM NIGMS NIH HHS; R01 NS089815 United States NS NINDS NIH HHS Contributed Indexing: Keywords: Adenosine triphosphate (ATP); Capillary electrophoresis (CE); Guanosine triphosphate (GTP); Immunoassay; Mass spectrometry (MS); Time-resolved luminescence (TRL) Substance Nomenclature: 86-01-1 (Guanosine Triphosphate) ; 8L70Q75FXE (Adenosine Triphosphate) ; K72T3FS567 (Adenosine) ; 12133JR80S (Guanosine) Entry Date(s): Date Created: 20230915 Date Completed: 20231026 Latest Revision: 20240210 Update Code: 20240210 PubMed Central ID: PMC10598090

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Homogeneous luminescent quantitation of cellular guanosine and adenosine triphosphates (GTP and ATP) using QT-Luc <superscript>GTP&ATP</superscript> assay.