Expected and Unexpected Results in the Separation of Distributions of Deuterosubstituted Multicharged Ions of Apamin and Its Complex

Abstract

This work is devoted to the analysis of the results obtained during the separation of the mass spectra of apamin and its complex after the deutero-hydrogen substitution of these molecules when leaving the electrospray ion source. The goal of the work was to determine the origin of the structural components identified in apamin and its complex. The separation method assumes that the substitution of labile hydrogen atoms with deuterium ones occurs independently, while charge carriers are retained independently too by various amino acid residues of a polypeptide with a specific structural form. This method also includes an indirect assessment of the contribution of natural isotopes to the measured data, without requiring knowledge of the elemental composition or other structural features of the biomolecule under study. Previously, we performed similar calculations by directly accounting for the contribution of natural isotopes. For apamin electrosprayed ions, after introducing a gas flow of ND₃ into the molecular ion reactor of the mass spectrometer, three main components were identified, with contributions of approximately 57, 39, and 5%. The new method yielded three components with similar contributions, which was anticipated. More noteworthy, though, and particularly relevant in various data collection and computation scenarios, is the paper’s description of the reliable identification of minor components with contributions of no more than 4%. Some of these components have almost identical formally found H/D distributions across different charges, remaining within or near the natural isotopic distribution range of apamin. This suggests that these components correspond to the structures of apamin ions and their complex in a solution without labile hydrogen atoms, such as during the formation of hydrogen bonds, or with a relatively small number of D-substituted hydrogen atoms, where protons carrying an ion charge do not participate in the exchange, particularly when more than two ion charges are present. Such a result was not anticipated in advance. This paper demonstrates the probable presence of three forms of apamin ions and its complex, each with distinct capacities for H/D exchange directly in the analyzed apamin solution. The limitations of the developed approach in decreasing the number of sites accepted to be involved in the exchange of hydrogen for deuterium, and in retaining charge carriers, are also discussed.