Gas-phase ion/ion covalent modification was coupled to ion mobility/mass spectrometry analysis to directly correlate the structure of gaseous ubiquitin to its solution structures with selective covalent structural probes. Collision cross section (CCS) distributions were measured prior to ion/ion reactions to ensure the ubiquitin ions were not unfolded when they were introduced to the gas phase. Ubiquitin ions were electrosprayed from aqueous and methanolic solutions yielding a range of different charge states that were analyzed by ion mobility and time-of-flight mass spectrometry. Aqueous solutions stabilizing the native state of ubiquitin generated folded ubiquitin structures with CCS values consistent with the native state. Denaturing solutions favored several families of unfolded conformations for most of the charge states evaluated. Gas-phase covalent labeling via ion/ion reactions was followed by collision induced dissociation of the intact, labeled protein to determine which residues were labeled. Ubiquitin 5+ and 6+ electrosprayed from aqueous solutions were covalently modified preferentially at the lysine 29 and arginine 54 residues, indicating that elements of secondary structure as well as tertiary structure were maintained in the gas phase. On the other hand, most ubiquitin ions produced in denaturing conditions were labeled at various other lysine residues, likely due to the availability of additional sites following methanol and low pH-induced unfolding. These data support the conservation of ubiquitin structural elements in the gas phase. The research presented here provides the basis for residue-specific characterization of biomolecules in the gas phase

<p dir="ltr">Metal ions are typically considered a nuisance for mass spectrometry, as they can introduce chemical noise and distribute an analyte’s signal into multiple peaks. In some cases however, metal ions in biological solutions are either necessary for biomolecular structures, or so ubiquitous in a sample’s native solution conditions that they are difficult to fully remove. In this work, the role of metal ions in biological analytes is explored. For analytes that require metal ions to maintain higher order structures, a mass spectrometry method was developed to determine whether a stable structure is formed from metal ion adducts, or if the metal ion adducts are nonspecifically bound. Electron transfer of these structures reveals complementary fragmentation information, with the added discovery of new radical fragmentation pathways. With mass spectrometry, specific ligand and metal ion affinities can even be determined for analytes at low enough concentrations. In addition to analytes that require metals, an exploration on unwanted metal ion adduction during the electrospray ionization process is shown via gas-phase ion/ion reactions. Observing how specific anionic ligands exchange metals with protons from proteins on a small and controlled scale gives a greater understanding of what solutions can lead to the cleanest results. In addition, this work shows the possibility of finding anionic ligands that will instead exchange protons with metal ions found on proteins. In the gas-phase, these experiments have a high degree of control, leading to a much greater understanding of how metal ions influence mass spectrometry samples.</p>