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 ( 2 H/ 1 H 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 δ 2 H 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. δ 2 H 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 δ 2 H and molecular-average δ 13 C 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 2 H-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.

Discovering Nature’s Fingerprints: Isotope Ratio Analysis on Bioanalytical Mass Spectrometers

“…Initial publications on model compounds such as oxyanions, amino acids, and organic acids demonstrate improved sensitivity in the nanomolar range. 24,66 They also highlight other promising methodological advances, such as CSIA, with simultaneous measurements of isotopes of several elements. Intramolecular Isotopocule Analysis.…”

“…Since in ESI-Orbitrap both ionization and ion detection are different from the classical gas-source IRMS, the new platform offers access to polar analytes at high sensitivity. Initial publications on model compounds such as oxyanions, amino acids, and organic acids demonstrate improved sensitivity in the nanomolar range. , They also highlight other promising methodological advances, such as CSIA, with simultaneous measurements of isotopes of several elements.…”

Position‐specific carbon isotope analysis of serine by gas chromatography/Orbitrap mass spectrometry, and an application to plant metabolism

“…Looking forward, a related instrument, the Q Exactive HF with an electrospray ionization source, is highly promising for isotope ratio analysis and has been studied in detail for other analytes including pure methionine, acetate, and inorganic ions. 14,[60][61][62] The greatest challenge for amino acid isotope analysis by this method is the lack of efficient fragmentation (e.g. for underivatized serine, the bond between C-2 and C-3 of serine does not fracture during electrospray ionization).…”

Position-Specific 2H/H Equilibrium Isotopic Fractionation Factors in Alkane, Alkene, and Aromatic Molecules: A Density Functional Theory Approach

“…Compound-specific H isotope data can be used to interpret the source(s) of petroleum. , Knowledge of the thermodynamics and kinetics of 2 H–H exchange at various types of C positions could facilitate such applications by enabling the evaluation of the imprint of isotopic exchange on molecular-averaged signatures. With the advent of multiple methods for position-specific deuterium isotope analyses, , researchers can benefit by modeling tools that enable them to evaluate data in comparison with calculated position-specific equilibrium signatures.…”