Electron spray ionization mass spectrometry and 2D 31P NMR for monitoring 18O/16O isotope exchange and turnover rates of metabolic oligophosphates

A new method was here developed for the determination of ¹⁸O-labeling ratios in metabolic oligophosphates, such as ATP, at different phosphoryl moieties (α-, β-, and γ-ATP) using sensitive and rapid electrospray ionization mass spectrometry (ESI-MS). The ESI-MS-based method for monitoring of ¹⁸O/¹⁶O exchange was validated with gas chromatography–mass spectrometry and 2D ³¹P NMR correlation spectroscopy, the current standard methods in labeling studies. Significant correlation was found between isotopomer selective 2D ³¹P NMR spectroscopy and isotopomer less selective ESI-MS method. Results demonstrate that ESI-MS provides a robust analytical platform for simultaneous determination of levels, ¹⁸O-labeling kinetics and turnover rates of α-, β-, and γ-phosphoryls in ATP molecule. Such method is advantageous for large scale dynamic phosphometabolomic profiling of metabolic networks and acquiring information on the status of probed cellular energetic system.