Probing the Position-Specific Hydrogen Isotopes of Acetate via ESI Orbitrap Mass Spectrometry

Abstract:

Acetate is a key intermediate in the deep biosphere. As an end product of fermentation and a substrate for anaerobic respiration, it is at the center of microbial carbon cycling, yet its sources and sinks are still poorly understood. The natural abundances of stable isotopes (e.g. ¹³C, D) in metabolites from the deep biosphere provide an in situ tool for tracing biogeochemical cycles. While carbon isotopes of acetate have been used extensively for these purposes, its hydrogen isotopes have not. The methyl-bound hydrogens on acetate originate from disparate sources and experience vastly different biological isotope fractionations depending on the metabolism that produced them. Therefore, we predict that the hydrogen isotope composition of acetate in the environment will depend strongly on the relative contributions from microbial metabolisms, making it a potentially useful tool for elucidating biogeochemical cycles in the deep biosphere. Previous techniques for measuring hydrogen isotopes of acetate have been limited in their applications due to millimolar sample concentration requirements. Here, we present a novel method for determining the position-specific hydrogen isotope composition of acetate at micromolar concentrations using an electrospray ionization (ESI) Orbitrap mass spectrometer. With a fast-fourier transform mass analysis, we resolve the monoisotopic, ¹³C and D isotopologues of acetate. We isolate these ions through a quadrupole mass filter and integrate their signals over the course of a measurement. This method produces both the carbon and hydrogen isotope composition of acetate. Furthermore, negative mode ESI removes the exchangeable carboxylic hydrogen, allowing for a position-specific analysis of the methyl-bound hydrogens. To correct for instrumental fractionation and place our measurements on the VSMOW and PDB scales, we created a series of acetate standards ranging in both δD and δ¹³C and measured their isotope compositions on Elemental Analyzers. Our studies of laboratory standards suggest that the ESI-Orbitrap achieves high precision with acetate solutions at micromolar concentrations, making it amenable to environmental samples. With this improved sensitivity, hydrogen isotopes could further constrain sources and sinks of acetate in the deep biosphere.