ION MOBILITY AND GAS-PHASE COVALENT LABELING STUDY OF THE STRUCTURE AND REACTIVITY OF GASEOUS UBIQUITIN IONS ELECTROSPRAYED FROM AQUEOUS AND DENATURING SOLUTIONS

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