Analysis of Oxygen-18 Labeled Phosphate to Study Positional Isotope Experiments Using LC-QTOF-MS
A method is proposed in this paper for the determination of oxygen-18 labeled phosphate so that positional isotope experiments using sensitive and rapid liquid chromatography–QTOF–mass spectrometry (LC-QTOF-MS) experiments can be carried out. The positional isotope exchange technique is a useful tool in understanding the mechanisms and kinetics of many enzyme-catalyzed reactions. Detection of the positions and concentration of these exchanged isotopes is the key. Gas chromatography–mass spectrometry (GC-MS) and nuclear magnetic resonance imaging are commonly used analytical techniques for measurement of 18O/16O, 31P and 15N isotope enrichment. Since these techniques either require a time-consuming derivatization step or have a limited sensitivity, an LC and accurate mass-based method for monitoring 18O/16O exchange was developed and compared with a standard GC-MS method. Our results showed that the LC-QTOF-MS method developed was not only as accurate as the standard GC-MS method, but also a sensitive and robust analytical platform for the simultaneous determination of isotope enrichment and the analysis of positional isotopes without chemical derivation. The LC-QTOF-MS method developed was successfully applied to the measurement of 18O/16O in the reversibility study of ATP hydrolysis by Lon proteases.