Scanning the isotopic structure of molecules by tandem mass spectrometry

Biomolecules generally exist as mixtures of diverse isotopologues that differ in the number and sites of rare-isotope substitutions. The exact proportion of isotopologues of a biomolecule may depend on the molecular, cellular, organismal and environmental factors involved in its biosynthesis, localization and consumption. Molecular isotopic structure can thus be a valuable tool to elucidate biochemical mechanisms and for the reconstruction of physiological, ecological and climatic processes. However, little information about this record is accessible by conventional methods of stable isotope chemistry. Here, we report an easy to implement mass spectrometric method that quantifies intramolecular isotope distributions and is specifically designed for use on samples containing low, natural abundances of the rare isotopes. Its essential feature is the use of a narrow initial mass selection window to isolate ions that are heavier due to the presence of one or more isotopic substitutions. This pre-selection greatly increases the relative proportions of the various rare-isotope substituted isotopologues. The selected ions are then fragmented, and within seconds to minutes the isotopic pattern of the fragment peaks reveals information about the intramolecular distribution of isotopes. This approach requires similar to 0.1-10 nanomole of analyte, which is about five orders of magnitude less than NMR. We demonstrate the ability to measure the site-specific isotope ratios of metabolites by resolving the C-13 content in the amino acid methionine among multiple non-equivalent carbon sites. This technique enables rapid origin assignments for a wide range of organic molecules and can be used for new applications of molecular isotopic structure in medicine and environmental sciences. (C) 2018 Elsevier B.V. All rights reserved.