Simultaneous, High-Precision Measurements of δ²H and δ¹³C in Nanomole Quantities of Acetate Using Electrospray Ionization-Quadrupole-Orbitrap Mass Spectrometry

Abstract

Stable hydrogen isotope compositions (²H/¹H ratios) have been an invaluable tool for studying biogeochemical processes in nature, but the diversity of molecular targets amenable to such analysis is limited. Here, we demonstrate a new technique for measuring δ²H of biomolecules via Orbitrap mass spectrometry (MS) using acetate as a model analyte. Acetate was chosen as a target molecule because its production and consumption are central to microbial carbon cycling, yet the mechanisms behind acetate turnover remain poorly understood. δ²H of acetate could provide a useful constraint on these processes; however, it remains uncharacterized in nature due to analytical challenges. Electrospray ionization (ESI)-Orbitrap MS circumvents these challenges and delivers methyl-specific H-isotope compositions of acetate with nanomole sensitivity, enough to enable analyses of environmental samples. This approach quantifies the methyl-specific δ²H and molecular-average δ¹³C of acetate simultaneously while achieving <3 and <0.5‰ uncertainty, respectively. Using optimized ionization and Orbitrap parameters, this level of precision is obtained within 15 min using only 15 nmol of acetate. As a demonstration of our analytical approach, we cultured three acetogenic bacteria and found a large ²H-fractionation between acetate and water (>310‰ depletion) associated with the Wood–Ljungdahl pathway, while fermentation expressed a muted (∼80‰) fractionation. With its high precision and sensitivity, Orbitrap MS is a promising tool for investigating these signals in nature after offline purification. Furthermore, the ESI-Orbitrap method presented here could be applied to other molecules amenable to ESI, including central metabolites and sugars, greatly expanding the molecular targets used in hydrogen isotope biogeochemistry.