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.
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.
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.
Analysis[edit]
Substrates need to be prepared and analyzed in a specific way to elucidate site specific isotope enrichments. This requires clean separation of the compound of interest from the original sample, which can require a variety of different preparatory chemistries. Once isolated, position-specific isotope enrichments can be analyzed with a variety of instruments, which all have different advantages and provide varying degrees of precision.
Enzymatic Reaction[edit]
To measure the kinetic isotope effects of enzymatic reactions, biochemists perform in vitro experiments with enzymes and substrates. The goal of these experiments is to measure the difference in the enzymatic reaction rates for the monoisotopic substrate and the substrate with one rare isotope. There are two popularly used techniques in these experiments: Internal competition studies and direct comparison experiments. Both measure position-specific isotope effects.
Direct Comparison[edit]
Direct comparison experiments are primarily used for measuring hydrogen/deuterium isotope effects in enzymatic reactions. The monoisotopic substrate and a deuterated form of the substrate are separately exposed to the enzyme of interest over a range of concentrations. The Michaelis-Menten kinetic parameters for both substrates are determined and the position-specific isotope effect at the site of deuteration is expressed as the ratio of the monoisotopic rate constant over the rare isotope rate constant.
Internal Competition[edit]
For isotopes like carbon and sulfur, the difference in kinetic parameters is too small, and the measurement precision too low, to measure an isotope effect by directly comparing the rates of the monoisotopic and rare isotope substrates. Instead, the two are mixed together using the natural abundance of stable isotopes in molecules. The enzyme is exposed to both isotopes simultaneously and its preference for the light isotope is analyzed by collecting the product of the reaction and measuring its isotope composition. For example, if an enzyme removes a carbon from a molecule by turning it into carbon dioxide, that carbon dioxide product can be collected and measured on an Isotope Ratio Mass Spectrometer for its carbon isotope composition. If the carbon dioxide has less 13C than the substrate mixture, the enzyme has preferentially reacted with the substrate that has a 12C at the site that is decarboxylated. In this way, internal competition experiments are also position-specific. If only the CO2 is measured, then only the isotope effect on the site of decarboxylation is recorded.
Chemical degradation[edit]
Before the advent of technologies that analyze whole molecules for their intramolecular isotopic structure, molecules were sequentially degraded and converted to CO2 and measured on an Isotope Ratio Mass Spectrometer, revealing position-specific 13C enrichments.
Ninhydrin Reaction[edit]
In 1961, Abelson and Hoering developed a technique for removing the carboxylic acid of amino acids using the ninhydrin reaction. This reaction converts the carboxylic acid to a molecule of CO2 which is measured via an Isotope Ratio Mass Spectrometer.
Ozonolysis Reaction[edit]
Lipids are of particular interest to stable isotope geochemists because they are preserved in rocks for millions of years. Monson & Hayes used ozonolysis to characterize the position-specific isotope abundances of saturated fatty acids, turning different carbon positions into carbon dioxide. Using this technique, they directly measured an isotopic pattern in fatty acids that had been predicted for years.
Preparatory Chemistry[edit]
Derivitization[edit]
In some cases, additional functional groups will need to be added to molecules to facilitate the other separation and analysis methods (i.e., chromatography). It is important to note, however, that derivatization is not ideal for site-specific analyses as it adds additional elements that must be accounted for in analyses.
Chromatography[edit]
Chromatography facilitates separation of distinct molecules within a mixture based on their respective chemical properties, and how those properties interact with the substrate coating the chromatographic column. This separation can happen “on-line,” during the measurement itself, or prior to measurements to isolate a pure compound. Gas and liquid chromatography have distinct advantages, based on the molecules of interest.
Instrumental Analysis[edit]
GC-IRMS and LC-MS[edit]
Initial measurements of position specific isotope enrichments were measured using isotope ratio mass spectrometry in which sites on a molecule were first degraded to CO2, the CO2 was captured and purified, and then the CO2 was measured for its isotope composition on an Isotope Ratio Mass Spectrometer (IRMS). Py-GC-MS was also used in these experiments to degrade molecules even further and characterize their intramolecular isotopic distributions. Both GC-MS and LC-MS are capable of characterizing position specific isotope enrichments in isotopically labelled molecules. In these molecules, 13C is so abundant that it can be seen on a mass spectrometer with low sensitivity. The resolution of these instruments can distinguish two molecules with a 1 Dalton difference in their molecular masses; however, this difference could arise from the addition of many rare isotope (17O, 13C, 2H, etc.). For this reason, mass spectrometers using quadrupoles or time-of-flight detection techniques cannot be used for measuring position-specific enrichments at natural abundances.
Spectroscopy[edit]
Laser spectroscopy can be used to measure isotope enrichments of gases in the environment. These measurements are fast and can reach relatively good precision (1-10 per mille), It is used to characterize environmental gas fluxes, and effects on these fluxes. This method is limited to measurement and characterization of gases.
Nuclear magnetic resonance (NMR)[edit]
Nuclear magnetic resonance observes small differences in molecular reactions to oscillating magnetic fields. As a result, NMR is able to identify site specific isotope enrichments within molecules.