Position-specific isotope analysis

Position-specific isotope analysis (PSIA), 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 nucleus, which changes the chemical mass of the element. Isotopes are found in varying natural abundance depending on the element; their abundance in distinct settings can vary from random distribution (i.e., stochastic distribution) due to environmental conditions that act on the mass variations differently. This process is called fractionation. Stable isotope analysis is the study of this variation.

Isotope abundances can vary across an entire substrate (i.e., “bulk” isotope variation), specific compounds within a substrate, or across positions within specific molecules. 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 shared a common limitation that they measured 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 chemistry studies these intramolecular variations, known as   “position-specific isotope” and “site-specific isotope” enrichments. It focuses on in 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. 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.

Isotopologues of ethanol (CH3CH2OH) with mass 47, corresponding to a single isotopic substitution. Isotopologues with a heavy isotope at different positions are called isotopomers. Ethanol has 2H- and 13C-isotopomers.

Theory[edit]

Dilution effect of site-specific enrichments. The 13C enrichment at the carboxylic acid site of an amino acid is less important as the structural resolution of the measurement is decreased to molecular average and bulk cell analyses.

Dilution effect of site-specific enrichments. The 13C enrichment at the carboxylic acid site of an amino acid is less important as the structural resolution of the measurement is decreased to molecular average and bulk cell analyses.

Isotope enrichment deviates from stochastic distribution, based on properties and environment the reaction is happening in. These deviations are meaningful. Kinetic isotope effects manifest in irreversible reactions, when one isotopologue is preferred in the transition state. Equilibrium isotope effects manifest in reversible reactions, when molecules can exchange freely to reach the lowest possible energy state.

Biological fractionation[edit]

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.

Mechanism[edit]

The position-specific isotope effect of an enzymatic reaction is expressed as the ratio of rate constants for a monoisotopic substrate and a substrate substituted with one rare isotope. A primary isotope effect is one in which the rare isotope is substituted where a bond is broken or formed. Secondary isotope effects occur on other positions in the molecule and are controlled by the molecular geometry of the transition state. These are generally considered to be negligible but do arise in certain cases, especially for hydrogen isotopes.

Unlike abiotic reactions, enzymatic reactions occur through a series of steps, including substrate-enzyme binding, conversion of substrate to product, and dissociation of enzyme-product complex. The observed isotope effect of an enzyme will be controlled by the rate limiting step in this mechanism. If the step that converts substrate to product is rate limiting, the enzyme will express its intrinsic isotope effect, that of the bond forming or breaking reaction.

Abiological fractionation[edit]

Like biotic molecules, position specific isotope enrichments in abiotic molecules can reflect the source of chemical precursors and synthesis pathways. For example, carbon in the interstellar medium and solar nebula partition into distinct states based on thermodynamic favorability. Measuring site-specific isotope enrichments of carbon from organic molecules extracted from carbonaceous chondrites can elucidate where each carbon atom comes from, and how organic molecules can be synthesized abiotically.

Another example of distinct site-specific fractionations in abiotic molecules is Fischer-Tropsch-type synthesis, which is thought to produce abiogenic hydrocarbon chains. Through this reaction mechanism, site enrichments of carbon would deplete as carbon chain length increases, and be distinct from site-specific enrichments of hydrocarbons of biological origins.

Techniques

The difficulty of position-specific isotope analyses relies on the fact that isotopomers have the same mass and are thus difficult to separate using conventional isotopic measurements. Several methods can be used to overcome these difficulties.

Chemical degradation: the molecule is degraded chemically into smaller fragments. Each of the fragment is separated and its isotopic composition analysed using a conventional isotope ratio mass spectrometer.

Pyrolysis: the sample is introduced into a pyrolysis furnace (which temperature is usually held between 600 °C and 1000 °C). Thermal cracking leads to the formation of fragments that are subsequently analyzed by IRMS.

High-resolution mass spectrometry: the molecule is fragmented by electron impact directly in the ion source of the spectrometer.

Nuclear Magnetic Resonance (NMR): each non-equivalent isotopomer of the molecule leads to a peak at a specific chemical shift. Their relative concentration is determined through the area of each peak. (Link: Isotopic analysis by nuclear magnetic resonance).

Density functional theory calculations: equilibrium isotope effects at the position-specific levels can be computed through DFT calculations for different temperatures.