Position-specific isotope analysis

Position-specific isotope analysis, also called site-specific isotope analysis, is a branch of isotope analysis aimed at determining the isotopic composition of a particular atom position in a molecule. Isotopes are elemental variants with different numbers of neutrons in their nuclei, thereby having different atomic masses. Isotopes are found in varying natural abundances depending on the element; their abundances in specific compounds can vary from random distributions (i.e., stochastic distribution) due to environmental conditions that act on the mass variations differently. These differences in abundances are called "fractionations," which are characterized via stable isotope analysis.

Isotope abundances can vary across an entire substrate (i.e., “bulk” isotope variation), specific compounds within a substrate (i.e., compound-specific isotope variation), or across positions within specific molecules (i.e., position specific isotope variation). Isotope abundances can be measured in a variety of ways (e.g., isotope ratio mass spectrometry, laser spectrometry, NMR, ESI-MS). Early analyses varied in technique, but were commonly limited by their ability to only measure average isotope compositions over molecules or samples. While this allows isotope analysis of the bulk substrate, it eliminates the ability to distinguish variation between different sites of the same element within the molecule. The field of position-specific isotope biogeochemistry studies these intramolecular variations, known as “position-specific isotope” and “site-specific isotope” enrichments. It focuses on position-specific isotope fractionations in many contexts, development of technologies to measure these fractionations and the application of position-specific isotope enrichments to questions surrounding biogeochemistry, microbiology, enzymology, medicinal chemistry, and Earth history. Position-specific isotope enrichments can retain critical information about synthesis and source of the atoms in the molecule. Indeed, bulk isotope analysis averages site-specific isotope effects across the molecule, and so while all those values have an influence on the bulk value, signatures of specific processes may be diluted or indistinguishable. While the theory of position-specific isotope analysis has existed for decades, new technologies exist now to allow these methods to be much more common. The potential applications of this approach are widespread, such as understanding metabolism in biomolecules, environmental pollutants in air, inorganic reaction mechanisms, etc. Clumped isotope analysis, a subset of position-specific isotope analysis, has already proven useful in characterizing sources of methane, paleoenvironment, paleoaltimetry, among many other applications. More specific case studies of position-specific isotope fractionation are detailed below.

Theory Stable isotopes do not decay, and the heavy and light isotope masses affect how they partition within the environment. Any deviation from a random distribution of the light and heavy isotopes within the environment is called fractionation, and consistent fractionations as a result of a particular process or reaction are called "isotope effects."

Isotope Effects Isotope effects are recurring patterns in the partitioning of heavy and light isotopes across different chemical species or compounds, or between atomic sites within a molecule. These isotope effects can come about from a near infinite number of processes, but most of them can be narrowed down into two main categories, based on the nature of the chemical reaction creating or destroying the compound of interest: (1) Kinetic isotope effects manifest in irreversible reactions, when one isotopologue is preferred in the transition state due to the lowest energy state. The preferred isotopologue will depend on whether the transition state of the molecule during a chemical reaction is more like the reactant or the product. Normal isotope effects are defined as those which partition the lighter isotope into the products of the reaction. Inverse isotope effects are less common as they preferentially partition the heavier isotope into the products. (2) Equilibrium isotope effects manifest in reversible reactions, when molecules can exchange freely to reach the lowest possible energy state. These variations can occur on a compound-specific level, but also on a position-specific level within a molecule. For instance, the carboxyl site of amino acids is exchangeable and therefore its carbon isotope signature can change over time and may not represent the original carbon source of the molecule.

Biological fractionation Chemical reactions in biological processes are controlled by enzymes that catalyze the conversion of substrate to product. Since enzymes can alter the transition state structure for reactions, they also change kinetic and equilibrium isotope effects. Placed in the context of a metabolism, the expression of isotope effects on biomolecules is further controlled by branch points. Different pathways of biosynthesis will use different enzymes, yielding a range of position specific isotope enrichments. This variability allows position-specific isotope measurements to discern multiple biosynthetic pathways from the same metabolic product. Biogeochemists use position specific isotope enrichments from amino acids, lipids, and sugars in nature to interpret the relative importance of different metabolisms.

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