Traditional versus isotopic fine structure HDX-MS of the [M + 2H] 2+of substance P. A. Isotope distributions of deuterated peptides (right) are shifted to heavier masses relative to the non-deuterated peptide (left). The monoisotopic isotopomer (green stars) appears depleted in the deuterated spectrum as it becomes labeled and consequently redistributed throughout the distribution. At typical resolving powers, natural abundance isotopomers and the isotopomers resulting from HDX that share the same nominal mass are not distinguished. B, C. In isotopic distributions where fine structure is resolved, deuterium-associated peaks (“pseudomonoisotopic” m/z’s corresponding to incorporation of 1, 2, or 3 deuterium are thus m/z 674.8745, m/z 675.3776, and m/z 675.8808, respectively) are distinct from the peaks corresponding to the presence of natural abundance isotopes such as the [M + 2H]2+ of substance P with exactly two 13C, which is present at m/z 675.3747.