Resolving Isotopic Fine Structure to Detect and Quantify Natural Abundance- and Hydrogen/Deuterium Exchange-Derived Isotopomers

Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) is used for analyzing protein dynamics, protein folding/unfolding, and molecular interactions. Until this study, HDX MS experiments employed mass spectral resolving powers that afforded only one peak per nominal mass in a given peptide’s isotope distribution, and HDX MS data analysis methods were developed accordingly. A level of complexity that is inherent to HDX MS remained unaddressed, namely, various combinations of natural abundance heavy isotopes and exchanged deuterium shared the same nominal mass and overlapped at previous resolving powers. For example, an A + 2 peak is comprised of (among other isotopomers) a two-2H-exchanged/zero-13C isotopomer, a one-2H-exchanged/one-13C isotopomer, and a zero-2H-exchanged/two-13C isotopomer. Notably, such isotopomers differ slightly in mass as a result of the ∼3 mDa mass defect between 2H and 13C atoms. Previous HDX MS methods did not resolve these isotopomers, requiring a natural-abundance-only (before ...