Ion Mobility and Gas-Phase Covalent Labeling Study of the Structure and Reactivity of Gaseous Ubiquitin Ions Electrosprayed from Aqueous and Denaturing Solutions

Abstract

Gas-phase ion/ion chemistry was coupled to ion mobility/mass spectrometry analysis to correlate the structure of gaseous ubiquitin to its solution structures with selective covalent structural probes. Collision cross section (CCS) distributions were measured to ensure the ubiquitin ions were not unfolded when they were introduced to the gas phase. Aqueous solutions stabilizing the native state of ubiquitin yielded folded ubiquitin structures with CCS values consistent with previously published literature. 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 conditions were covalently modified preferentially at the lysine 29 and arginine 54 positions, indicating that elements of three-dimensional 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 pHinduced 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.

Identification of Phosphatidylcholine Isomers in Imaging Mass Spectrometry Using Gas-Phase Charge Inversion Ion/Ion Reactions

“…Herein, we demonstrate the ability of ion/ion reactions to alter the ion type in the gas-phase following MALDI ionization in order to provide enhanced chemical structural information for lipids generated directly from tissue surfaces. The McLuckey group and others have previously described a wide variety of ion/ion reactions for use in studying protein conformations, [39][40][41][42][43][44] peptide sequences, [45][46][47][48][49][50][51][52][53][54] oligonucleotide sequences, 55,56 and, more recently, lipid structures. [57][58][59][60] Gas-phase ion/ion reactions are fast, efficient, and specific, 61,62 making them ideally suited for use in imaging mass spectrometry.…”

Gas-phase ion/ion chemistry for structurally sensitive probes of gaseous protein ion structure: Electrostatic and electrostatic to covalent cross-linking

“…We have also previously published on the gas phase ion/ion reaction between ubiquitin and sulfo-benzoyl HOAt, where the sulfonate group first attaches to an accessible protonated site, followed by a covalent reaction with a nearby nucleophilic residue and the loss of the HOAt leaving group. 23 In addition, sulfo-benzoyl HOAt and sulfo-NHS acetate sulfonates likely have similar reactivity with protonated side chains. Since the through-bond distance from the reactive carbonyl carbon to the sulfonate oxygens in the reagent is approximately 6.4 Å, this provides a spatial constraint for the possible sites for covalent modification (Figure S5) Supplementary Table 2B).…”

“…cell (e-MSion, Corvallis, OR). Instrumental details for the ion/ion reactions 24,37 and ExD cell 33 have been described previously. The NanoLockspray source was used with proteins infused through the sample sprayer and anionic reagents through the reference sprayer..…”

“…This lower pressure regime was used to maintain conditions in the trap cell conducive to stable ion/ion product formation and charge state-based separation of the ion/ion products, as previously reported. 37 A ramping traveling wave velocity of 600 -3400 m/s over a full IMS cycle and wave height of 40 V were used in the mobility cell. After IM separation, the ions were subjected to ECD fragmentation (using 3.5 eV electrons) in the ExD cell (detailed voltage settings included in Table 1 of supplementary info) before mass analysis in the time-of-flight (TOF) mass spectrometer set to 'Resolution' mode.…”

“…22 Gas-phase ion/ion reactions have also been used to report on threedimensional structure of protein cations formed from native-like or denaturing solutions. [23][24][25] A combination of experimental 4,7,[26][27][28][29] and computational studies [30][31] have shown that under carefully-controlled experimental conditions, the solution structures of electrosprayed peptides and proteins is conserved upon electrospray ionization, making methods for probing gas-phase three-dimensional structures of proteins an extremely valuable addition to structural biology techniques.…”