Ion Mobility of Proteins in Nitrogen Gas: Effects of Charge State, Charge Distribution, and Structure

Ion mobility is emerging as a rapid and sensitive tool for structural characterization. Collision cross-section (Ω) values determined using ion mobility are often compared to values calculated for candidate structures generated through molecular modeling. Several methods exist for calculating Ω values, but the trajectory method explicitly includes contributions from long-range, ion–neutral interactions. Recent implementations of the trajectory method have significantly reduced its expense and have made applications to proteins far more tractable. Here, we use ion mobility experiments and trajectory method calculations to characterize the effects of charge state, charge distribution, and structure on the ion mobility of proteins in nitrogen gas. These results show that ion-induced dipole interactions contribute significantly to the Ω values of these ions with nitrogen gas, even for the modestly charged ions commonly observed in native mass spectrometry experiments. Therefore, these interactions contribute significantly to the values measured in most structural biology and biophysics applications of ion mobility using nitrogen gas. Comparisons between the reciprocal mobilities of protein ions in helium gas and in nitrogen gas show that there are significant, noncorrelated differences between these values. As a consequence, it is challenging to estimate the errors associated with interconverting between helium- and nitrogen-based mobilities without extensive characterization in both gases, even for ions of proteins with similar sequences. Therefore, we recommend reporting Ω and mobility values that are based on the predominant gas present in the separation and applying additional caution when comparing results from mobility experiments performed using different gases

Comprehensive Analysis of Gly-Leu-Gly-Gly-Lys Peptide Dication Structures and Cation-Radical Dissociations Following Electron Transfer: From Electron Attachment to Backbone Cleavage, Ion–Molecule Complexes, and Fragment Separation

Experimental data from ion mobility measurements and electron transfer dissociation were combined with extensive computational analysis of ion structures and dissociation energetics for Gly-Leu-Gly-Gly-Lys cations and cation radicals. Experimental and computational collision cross sections of (GLGGK + 2H)²⁺ ions pointed to a dominant folding motif that is represented in all low free-energy structures. The local folding motifs were preserved in several fragment ions produced by electron transfer dissociation. Gradient optimizations of (GLGGK + 2H)^+• cation-radicals revealed local energy minima corresponding to distonic zwitterionic structures as well as aminoketyl radicals. Both of these structural types can isomerize to low-energy tautomers that are protonated at the radical-containing amide group forming a new type of intermediates, −C^•O^–NH₂⁺– and −C^•(OH)­NH₂⁺–, respectively. Extensive mapping with B3LYP, M06-2X, and MP2­(frozen core) calculations of the potential energy surface of the ground doublet electronic state of (GLGGK + 2H)^+• provided transition-state and dissociation energies for backbone cleavages of the N–C_α and amide C–N bonds leading to ion–molecule complexes. The complexes can undergo facile prototropic migrations that are catalyzed by the Lys ammonium group and isomerize enolimine <i><b>c</b></i>-type fragments to the more stable amide tautomers. In contrast, interfragment hydrogen atom migrations in the complexes were found to have relatively high transition energies and did not compete with fragment separation. The extensive analysis of the intermediate and transition-state energies led to the conclusion that the observed dissociations cannot proceed competitively on the same potential energy surface. The reactive intermediates for the dissociations originate from distinct electronic states that are accessed by electron transfer