Foundations of Molecular 'Isotomics'

ACCOMPLISHMENTS1. What are the major goals of the project?This report describes research conducted during the 2016-2023 performance period for a program that aimed to develop the enabling technologies, methods and computa�onal tools for the study of intramolecular isotopic structures of natural and synthe�c materials, and to demonstrate these tools through useful applica�ons to geochemistry and forensics.The goals of the program, as defined in the ini�al proposal that led to its launch, were to inves�gate the isotopic structures of amino acids formed under a variety of condi�ons, to recognize dis�nc�ve 'fingerprints' of specific forma�on mechanisms, to elucidate the mechanisms of forma�on where they are incompletely known, and to examine how important biogeochemical reac�ons manifest in the isotopic structures of amino acids.Ques�ons we intended to address include:• Is it possible to observe mul�ple proper�es of the isotopic structures of amino acids on samples of a size that can be recovered from natural systems (sub-micromole)?No previous study of the isotopic structures of organic compounds has demonstrated this capability, so success will cons�tute a major advance in technological capability.• How do the isotopic structures of amino acids respond to controlled physical, chemical and biological reac�ons?• Can we recognize isotopic fingerprints that clearly dis�nguish biogenic from abiogenic amino acids?• How do the isotopic composi�ons of amino acids differ among different types of organisms?• Can we tell the difference between amino acids grown under different environmental condi�ons;for example, can we dis�nguish between amino acids from an autotroph growing at very different temperatures, such as the deep subsurface vs. shallow ground waters?Our work was organized into the following categories of tasks: Technologies and Methods We established mass spectrometric techniques to enable the analysis of mul�ple site-specific and clumped isotope proper�es of sub-micromolar samples of amino acids, with precision of ~1 ‰.Our ini�al focus was on alanine, and included measurements of its three singly-13 C-subs�tuted species, abundances of two 13 C-13 C and one 13 C-14 N 'clumped' species, the D/H ra�o of the non-exchangeable hydrogen in its methyl group, as well as a direct mass spectrometric measurement of the 15 N content of its one N site (i.e., all 8 of these proper�es will be returned by a single mass spectrometric measurement).A 'stretch goal' of this work was to translate these capabili�es to other amino acids.Controlled Experiments: We used these methods to observe frac�ona�ons of alanine isotopologues resul�ng from controlled laboratory procedures, where it should be rela�vely straigh�orward to reach a mechanis�c interpreta�on of our results.These experiments included both physical processes, such as solva�on and evapora�on, chemical processes, such as Strecker synthesis, and biological processes, such as consump�on and produc�on of alanine by cultured microorganisms.Studies of Natural Amino Acids: We used these methods to survey the isotopic structures of diverse biogenic and some non-biogenic amino acid sources.This work reproduced and expand on the pioneering data from Abelson and Hoering, 1961, and will include comparisons with alanines known or suspected to have been formed by natural abiological syntheses, and comparisons among disparate forms of life (e.g., extremophiles vs. common marine cyanobacteria).The findings of this ini�al finding period (see below) included the discovery that Orbitrap based mass spectrometers provided a uniquely powerful path to generalized study of molecular isotope structures; as a result, our goals in the second funding period (2019-2023) shi�ed to focus on this specific set of technologies.Specific proposed tasks and approximate schedule for this more recent extended performance period were : Year 1 (2019-2020): -Advance capabili�es for Orbitrap based analysis of small and mixed samples -Study accuracy of FT-ICR analyses of carbon isotope ra�os of pep�des (performed at PNNL) -Construct models of alanine and pyruvate par��on func�on ra�os, including solvated species, at higher levels of theory (including path integral methods).-Survey isotopic structures of representa�ve methionines in the human food supply -Devise and test methods for analysis of C and H isotope structure of phytane -Outline model of molecular isotopic structures in reac�on networks and construct data base of experimental and first-principles constraints on par��on func�on ra�os and KIEs -Complete documents that provide instruc�on on methods of Orbitrap based isotopic analysis and associated data processing and triaging.-Devise and test methods for isotopic analysis of DMMP and Dichlor by Q Exac�ve GC, peak trapping methods Year 2 (2020-2021): -Compare isotopic structures of Archaeal and Bacterial phytane -Devise and test methods for analysis of C and H isotope structures of steranes and hopanes -Study Orbitrap based methods of clumped isotope analysis, including tests of the ability to drive precision down to levels below 0.1 ‰ -Conduct experiments on 13 C labeled alanine/pyruvate interconversion to confirm reversible interconversion of reac�ve C sites.-Complete and test semi-empirical models of par��on func�on ra�os and KIE's for integrated reac�on network model -Survey isotopic structures of representa�ve methionines in the human food supply -Beta test Orbitrap based protocols for isotopic analysis, using visitors to Caltech and collaborators at Thermo-Fisher Bremen, PNNL, Penn State, and the European doping agency as testers.-Begin survey of isotopic structures of DMMP and Dichlor -Assemble parts (e.g., pneuma�c replacement valves) needed for automa�on of peak trapping analysis; prepare Labview script to execute those measurements.Year 3 (2021-2022):-Test the abili�es of our integrated predic�ve model of isotopic structures to match independent constraints from well-studied reac�ons, such as the Diels-Alder chemistry and hemoly�c cleavage of hydrocarbons.-Distribute revised protocols for Orbitrap based isotopic analysis -Examine the C and H isotope structures of steranes and hopanes from culture.-Develop and test new peak trapping reservoirs and similar devices designed for conveying samples from preparatory GC apparatus to our analy�cal apparatus -Construct and experimentally test models of isotope effects associated with DMMP and Dichlor synthesis -Develop and test methods for automated peak trapping and analysis method on Q Exac�ve GC The goals and schedule of work we proposed for the 2019/2020 funding year were impacted by two developments in laboratory or group resources: • The laboratories supervised by the PI funded and quickly purchased and installed a second Orbitrapbased mass spectrometry system to complement the one that has served as the basis of most prior work in our program.The new system, a Thermo Fisher Q Exactive HF with integrated HPLC and

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