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Sessions, Alex L.

Publications and source records attributed to Sessions, Alex L..

Foundations of Molecular 'Isotomics'

The naturally occurring rare isotopes are versions of common elements, such as hydrogen, carbon and oxygen, that contain a larger than usual number of neutrons in their atomic nuclei and therefore are higher in mass than the common atoms of that element. Isotopes exist for most elements and are found in most natural and synthetic materials, but are uneven in their distribution because chemical and physical processes are isotope-selective (e.g., a chemical reaction may proceed more rapidly for one isotope than for another). For this reason, abundances of isotopes in a material of interest can provide a record, or ‘signature’ of various features of that material’s origin and history. These signatures have been used in the geo, life, chemical and physical sciences in a wide variety of ways over close to 8 decades. However, many such applications struggle to reach unique interpretations of isotopic data because multiple factors combine to control a given sample’s overall isotopic content. That is, the factors controlling isotopic content are too numerous and complex to fully constrain from a simple measurement of a material’s isotope abundances. However, the distribution of isotopes within materials, at molecular scales potentially provides a vastly larger number and diversity of constraints on the chemical and physical processes that comprise a material’s history. The rare isotopes may be concentrated into one atomic position in a molecule relative to another, some proportion of molecules in a sample may contain two or more rare isotopes, and those multiply-isotope-substituted forms of molecules may also have uneven distributions of those isotopes across individual atomic sites. For these reasons, even small, seemingly simple molecules, such as sugars, amino acids or drug compounds, actually exist in a vast number of isotopically unique forms (often millions or more), and each one of those forms is in some sense an independent ‘vote’ on that sample’s history. This project has focused on opening this rich archive of information by enabling the creation of routinely and widely applicable ways of measuring and interpreting isotopic structures of molecules. This work has included the development of core technologies and analytical methods, advancing fundamental understanding of the physical and chemical properties of isotopic versions of molecules, and conducting proof of concept studies of illustrative geochemical, cosmochemical and forensic problems in order to show how these technologies, methods and principles come together to solve problems in new ways. A key to the success of this project was the adaptation of ‘Fourier transform mass spectrometry’ (FTMS) to the task of precisely measuring proportions of the rare, naturally occurring isotopic forms of molecules. FTMS is a highly specialized form of mass spectrometry that traps ions within magnetic or electrostatic cavities and, effectively, ‘listens’ (through registering of subtle electrical signals) to the harmonic signals they make while rapidly orbiting within those cavities. These signals have periods that are a function of their mass and strength (or ‘loudness’) that is proportional to their abundances. Thus, these signals constrain relative amounts of molecules that differ in their mass due to various isotopic substitutions. This technology has been essential to the identification of organic molecules in the life, chemical and environmental sciences for over 4 decades, but generally has lacked the control, stability and precision to meaningfully measure rare isotope forms of molecules. This project’s most fundamental contribution has been to modify FTMS, both in terms of hardware and methods, to enable such measurements. The raw data of molecular isotopic structure is tremendously voluminous and complex, so another important activity of this project has been developing the theoretical and data-science tools needed to interpret the data generated by this new form of isotopic measurement. A particularly challenging part of this task has been predicting molecular isotopic structure, as only through the comparison of measurements with predictions can we make progress on hypothesis driven research questions. We have attacked this this prediction task through a combination of first-principles chemical-physics models of the effects of isotope substitution on molecule properties and data-science models that permit us to generalize that chemical physics to cases that have not yet been studied by detailed chemical physics theory. The proof of concept applications we have pursued over the course of this study include biological reactions of amino acids and other biomolecules, non-biological synthesis of organic molecules in extra-terrestrial settings such as meteorites, petroleum geoscience questions concerning the origin and evolution of natural gas, oil and kerogen compounds, and forensic questions such as the sourcing of chemical weapons. The successes of these applications have laid the groundwork for the next phase of this field’s development, which will include larger scale and more ambitious studies of molecular isotopic structure as a means of diagnosing human diseases, such as cancer, and reconstructing detailed interpretations of the origin and evolution of organic molecules in modern and geological environments.

Cesar, Jaime↗

High-dimensional isotomics, part 2: Observations of over 100 constraints on methionine's isotome

The abundances of different isotopic forms of a compound, or isotopologues, will vary based on its physical and chemical history. The number of isotopologues increases combinatorically with the size of a molecule, and even small molecules such as amino acids have thousands of potentially observable isotopic variants. However, due to the analytical challenges of separating and observing isotopologues, only a few dimensions of isotopic diversity are routinely measured. Overcoming these challenges requires both an experimental method to observe many isotopic properties and a theoretical framework for interpreting these experiments. In Part 1, we presented such a theoretical framework; here, we demonstrate an experimental method, which we apply to methionine. Our approach uses a Q Exactive HF Orbitrap to perform several “M + N experiments”, where a sample is ionized, a subset of its isotopologues with cardinal mass N daltons greater than the unsubstituted isotopologue is selected and fragmented, and the proportions of all detectable isotopic forms of those fragment ions are quantified. We perform M + 1, M + 2, M + 3, and M + 4 experiments of a methionine sample and standard where the sample has a 100 ‰ enrichment of 13C at the methyl carbon relative to the natural 13C abundance at that position in the standard, and is otherwise identical to the standard. We observe isotopic forms of 8 fragment ion species for each version of the M + N experiment. With the assistance of a forward model of expected mass spectra, we identify isotopic peaks for each fragment ion based on observed mass and abundance, screen these for data quality, and quantify abundances for 146 unique isotopic peaks at precisions of ≈ 0.3–3 ‰. We present our direct observations and use them to reconstruct the concentrations of 19 individual singly, doubly, and triply-substituted isotopologues; doing so gives fewer constraints and broader error bars than working with the direct observations, but may be more interpretable for some applications. We also examine possibilities for measuring additional peaks, which are primarily limited by the detection limit of the Orbitrap-IRMS method. We then suggest some possible uses of our direct measurements for chemical forensics and hypothesis testing. Furthermore, our results demonstrate the diversity of isotopic constraints currently observable and interpretable for organic molecules.

58 GEOSCIENCES↗

Position-specific carbon isotopes of Murchison amino acids elucidate extraterrestrial abiotic organic synthesis networks

The Murchison meteorite is a well-studied carbonaceous chondrite with relatively high concentrations of amino acids thought to be endogenous to the meteorite, in part because they are characterized by carbon isotope (δ 13 C) values higher than those typical of terrestrial amino acids. Past studies have proposed that extraterrestrial amino acids in the Murchison meteorite could have formed by Strecker synthesis (for α-amino acids), Michael addition (for β-amino acids), or reductive amination, but a lack of constraints have prevented confident discrimination among these possibilities, or assignment of specific formation pathways to each of several specific amino acids. Position-specific carbon isotope analysis differentiates amongst these mechanisms by relating molecular sites to isotopically distinct carbon sources and by constraining isotope effects associated with elementary chemical reactions. Prior measurements of the position-specific carbon isotopic composition of α-alanine from the Murchison CM chondrite demonstrated that alanine’s high δ 13 C VPDB value is attributable to the amine carbon (δ 13 C VPDB = +142 ± 20‰), consistent with Strecker synthesis drawing on 13 C-rich carbonyl groups in precursors (Chimiak et al., 2021). Here, we measured the δ 13 C composition of fragment ions generated by electron impact ionization of derivatized α-alanine, β-alanine, and aspartic acid from Murchison via gas chromatography-Fourier transform mass spectrometry. α-Alanine’s amine carbon yielded δ 13 C VPDB = +109 ± 21‰, which is consistent with the previously measured value and with formation from 13 C-rich precursors. β-Alanine’s amine carbon presents a lower δ 13 C VPDB = +33 ± 24‰, which supports formation from 13 C-rich precursors but potentially via a Michael addition mechanism rather than Strecker synthesis. Aspartic acid’s amine carbon has δ 13 C VPDB = -14 ± 5‰, suggesting synthesis from precursors distinct from those that generated the alanine isomers. Further, these measurements indicate that Murchison amino acids are a mixture of compounds made from different synthesis mechanisms, though some subsets likely drew on the same substrates; this conclusion highlights the complexity of extraterrestrial organic synthesis networks and the potential of emerging methods of isotope ratio analysis to elucidate the details of those networks.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Sulfur cycling at natural hydrocarbon and sulfur seeps in Santa Paula Creek, CA

Biogeochemical cycling of sulfur is relatively understudied in terrestrial environments compared to marine environments. However, the comparative ease of access, observation, and sampling of terrestrial settings can expand our understanding of organisms and processes important in the modern sulfur cycle. Furthermore, these sites may allow for the discovery of useful process analogs for ancient sulfur-metabolizing microbial communities at times in Earth's past when atmospheric O 2 concentrations were lower and sulfide was more prevalent in Earth surface environments. Here, we identified a new site at Santa Paula Creek (SPC) in Ventura County, CA—a remarkable freshwater, gravel-bedded mountain stream charged with a range of oxidized and reduced sulfur species and heavy hydrocarbons from the emergence of subsurface fluids within the underlying sulfur- and organic-rich Miocene-age Monterey Formation. SPC hosts a suite of morphologically distinct microbial biofacies that form in association with the naturally occurring hydrocarbon seeps and sulfur springs. We characterized the geology, stream geochemistry, and microbial facies and diversity of the Santa Paula Creek ecosystem. Using geochemical analyses and 16S rRNA gene sequencing, we found that SPC supports a dynamic sulfur cycle that is largely driven by sulfide-oxidizing microbial taxa, with contributions from smaller populations of sulfate-reducing and sulfur-disproportionating taxa. This preliminary characterization of SPC revealed an intriguing site in which to study geological and geochemical controls on microbial community composition and to expand our understanding of sulfur cycling in terrestrial environments.

58 GEOSCIENCES↗

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

Position-specific 13 C/ 12 C ratios within amino acids remain largely unexplored in environmental samples due to methodological limitations. We hypothesized that natural-abundance isotope patterns in serine may serve as a proxy for plant metabolic fluxes including photorespiration. Here we describe an Orbitrap method optimized for the position-specific carbon isotope analysis of serine to test our hypothesis and discuss the generalizability of this method to other amino acids. Position-specific carbon isotope ratios of serine were measured using a Thermo Scientific™ Q Exactive™ GC Orbitrap™. Amino acids were hydrolyzed from Arabidopsis biomass, purified from potential matrix interferences, and derivatized alongside standards. Derivatized serine (N,O-bis(trifluoroacetyl)methyl ester) was isolated using gas chromatography, trapped in a reservoir, and purged into the electron ionization source over tens of minutes, producing fragment ions containing different combinations of atoms from the serine-derivative molecule. The 13 C/ 12 C ratios of fragments with monoisotopic masses of 110.0217, 138.0166, and 165.0037 Da were monitored in the mass analyzer and used to calculate position-specific δ 13 C values relative to a working standard. This methodology constrains position-specific δ 13 C values for nanomole amounts of serine isolated from chemically complex mixtures. The δ 13 C values of fragment ions of serine were characterized with ≤1‰ precisions, leading to propagated standard errors of 0.7–5‰ for each carbon position. Position-specific δ 13 C values differed by up to ca 28 ± 5‰ between serine molecules hydrolyzed from plants grown under contrasting pCO 2 , selected to promote different fluxes through photosynthesis and photorespiration. The method was validated using pure serine standards characterized offline. Here this study presents the first Orbitrap-based measurements of natural-abundance, position-specific carbon isotope variation in an amino acid isolated from a biological matrix. We present a method for the precise characterization of isotope ratios in serine and propose applications probing metabolism in plants. We discuss the potential for extending these approaches to other amino acids, paving the way for novel applications.

59 BASIC BIOLOGICAL SCIENCES↗

Practical considerations for amino acid isotope analysis

Over the last few decades, isotopic analysis of amino acids at the compound- and position-specific levels has been rapidly advancing across diverse fields. As these techniques progress, evaluation of isotopic fractionation associated with sample workup is essential. This critical review of analytical methods through the lens of isotope geochemistry provides a benchmark for researchers across disciplines seeking to make compound- and position-specific amino acid isotope measurements. We focus on preparation, acid hydrolysis, clean-up, derivatization, separation, and C, H, N, and S isotope measurement. Despite substantial customizability across these steps, the following general recommendations should maximize recovery while minimizing isotopic fractionation. Samples should be freeze-dried and stored anoxically at ≤ –20 °C prior to conventional acid hydrolysis (6N HCl, 110 °C, 20–24 h, anoxic), which suffices for many residues. Both gas and liquid chromatographic (GC and LC, respectively) techniques are well-established and separate about 15 amino acids; LC bypasses the need for derivatization, while GC provides higher sensitivity. Furthermore, when derivatization is needed, n-acetyl and alkoxycarbonyl esters provide the most reproducible C isotope ratios. For compound-specific analyses, online GC–IRMS and LC–IRMS systems offer the easiest workflow, but EA–IRMS enables potential multi-element isotope analysis. Emerging techniques like high-resolution mass spectrometry are also promising for multi-element analysis and recover position-specific isotopic information. Looking forward to the next decade of innovation, isotope geochemists and ecologists can improve amino acid isotope analysis by focusing on streamlining multi-element analysis and standardizing calibration practices across laboratories.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗