A guide to precise measurements of isotope abundance by ESI-Orbitrap MS

Stable isotopes of carbon, hydrogen, nitrogen, oxygen and sulfur are widespread in nature. Nevertheless, their relative abundance is not the same everywhere. This is due to kinetic isotope effects in enzymes and other physical principles such as equilibrium thermodynamics. Variations in isotope ratios offer unique insights into environmental pollution, trophic relationships in ecology, metabolic disorders and Earth history including climate history. Although classical isotope ratio mass spectrometry (IRMS) techniques still struggle to access intramolecular information like site-specific isotope abundance, electrospray ionization-Orbitrap mass spectrometry can be used to achieve precise and accurate intramolecular quantification of isotopically substituted molecules ('isotopocules'). This protocol describes two procedures. In the first one, we provide a step-by-step beginner's guide for performing multi-elemental, intramolecular and site-specific stable isotope analysis in unlabeled polar solutes by direct infusion. Using a widely available calibration solution, isotopocules of trifluoroacetic acid and immonium ions from the model peptide MRFA are quantified. In the second approach, nitrate is used as a simple model for a flow injection routine that enables access to a diverse range of naturally occurring isotopic signatures in inorganic oxyanions. Each procedure takes 2-3 h to complete and requires expertise only in general mass spectrometry. The workflows use optimized Orbitrap IRMS data-extraction and -processing software and are transferable to various analytes amenable to soft ionization, including metabolites, peptides, drugs and environmental pollutants. Optimized mass spectrometry systems will enable intramolecular isotope research in many areas of biology. Carbon, hydrogen, nitrogen, oxygen and sulfur have naturally occurring stable isotopes. Variations in their relative distribution within molecules can provide information relevant to environmental biology, metabolism, food and drug origins and climate history.This protocol describes how to use electrospray ionization-Orbitrap mass spectrometry to achieve precise and accurate molecular quantification of isotopically substituted molecules ('isotopocules'). It serves as a citable reference for optimized Orbitrap IRMS data-extraction and -processing software. Isotope ratio mass spectrometry as described in this protocol can be used to determine natural variation in the abundance of stable isotopes in individual compounds to provide information relevant to metabolism, ecology or climate change.