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Published January 18, 2022 | Supplemental Material
Journal Article Open

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.

Additional Information

© 2021 American Chemical Society. Received: September 23, 2021; Accepted: December 20, 2021; Published: December 30, 2021. The authors gratefully acknowledge Jared Leadbetter and Victoria Orphan (Caltech) for use of their laboratory spaces, Nathan Dalleska (Environmental Analysis Center, Caltech) for expertise in the ion chromatography system, Volker Müller (Goethe University, Frankfurt, Germany) for providing both strains of autotrophic acetogens, Tim Csernica, Sarah Zeichner, and Elise Wilkes for providing data analysis scripts, and Fenfang Wu (Caltech) for assistance with EA-IRMS measurements. This work was made possible by an NSF Graduate Research Fellowship DGE-1745301 (to E.P.M.) and the NASA Astrobiology Institute grant # 80NSSC18M0094 (to J.M.E. and A.L.S.). Author Contributions. E.P.M initiated the project and performed ESI-Orbitrap and IC experiments. E.P.M. and G.M.W. grew bacterial cultures. P.E.S. carried out TC-EA-IRMS experiments. E.P.M., A.L.S., and J.M.E interpreted experimental data. All authors contributed to writing the manuscript. The authors declare no competing financial interest.

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Created:
September 22, 2023
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October 23, 2023