Uneven Distribution of Oxygen Lability within Phosphatidylserine Evaluated with Oxygen Exchange and Tandem Mass Spectrometry

Abstract

Understanding the mechanism of isotope exchange between biomolecules and solvent molecules has very important implications in two areas of life sciences, namely, the studies of biomolecular dynamics using the exchange of labile atoms as a structural probe and the investigation of metabolic pathways using stable isotope-labeled (SIL) compounds. While the former has benefited greatly from the fast exchange of heteroatom-bound hydrogens with water molecules, several other elements have been shown to exhibit fast-to-moderate exchange kinetics with the solvent molecules, raising the hopes that they can also be used as structural probes in isotope exchange reactions. Conversely, significant isotope exchange between SIL compounds and the solvent molecules in metabolic studies would be highly detrimental vis-à-vis the reliability of quantitative (and in extreme cases also qualitative) aspects of such investigations. We use phosphatidylserine 16:0/16:0 (PS) as a model compound to probe catalyst-free oxygen exchange (¹⁶O/¹⁸O) with water in a site-specific fashion using tandem mass spectrometry (MS/MS) as a readout. All three oxygen-containing functionalities are targeted: the carboxylate end-group, the phosphate linker, and the fatty acids’ acyl ester groups (nonbridging oxygen atoms). Oxygen exchange within both the carboxylic moiety and the acyl ester groups is slow under the physiologically relevant conditions but is dramatically accelerated in acidic media at elevated temperatures. In contrast, exchange of the oxygen atoms within the phosphate linker remains below the limit of detection under all conditions tested in this work. While these results may be viewed as disappointing vis-à-vis the prospects of using oxygen exchange to study phospholipid dynamics (e.g., to quantify PS localization on the outer membrane leaflet upon cell activation or apoptosis), they are both reassuring and helpful in the context of studies relying on stable isotopes as tracers of metabolic processes. Indeed, the uneven distribution of oxygen lability across three different functionalities, as well as the identification of conditions that result in oxygen exchange acceleration, will be invaluable for both designing the SIL compounds and selecting the sample-handling conditions that eliminate the possibility of “isotope leaching” in the studies of metabolic pathways.