Analysis of intramolecular carbon isotope distributions in alanine by electrospray ionization Orbitrap mass spectrometry

The ability to detect intramolecular isotopic differences within a single molecule can answer questions about molecule synthesis and alteration across numerous scientific fields. Until recently, intramolecular (i.e., position-specific isotope analysis, PSIA) isotope measurements were laborious, requiring large amounts of analyte or specialized instrumentation. Orbitrap™ mass spectrometers are capable of fragmenting molecules and have the high mass resolution needed to constrain position-specific isotopic differences among the resulting fragment ions. Orbitrap mass spectrometers with electrospray ionization accurately measured the molecular average isotope composition of acetate, nitrate, sulfate, phosphate, and the amino acid methionine, as well as the position-specific isotopic structure of methionine. Here, we document the ability of this method to measure the position-specific carbon isotope structure of the amino acid alanine. Data include measurements of ¹³C-enriched materials to assign specific atoms in fragments to the original molecular structure and detect any recombination of atoms in resultant fragments. We further demonstrate high-precision intramolecular isotope analyses for standards with independently determined position-specific carbon isotope compositions. Isotope data from ESI-Orbitrap-MS agrees with values obtained using gas source isotope ratio mass spectrometry, giving further confidence to this novel approach to PSIA. The carbon isotope analyses by Orbitrap-MS were rapid and required ∼5 μg of analyte to obtain both molecular average and position-specific values in triplicate with precision ≤1‰.