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-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.

Keywords: covalent labeling; ion mobility; ion/ion reactions; native mass spectrometry.

Figure 1.

Intensity normalized arrival time distributions (ATDs) of ubiquitin 5⁺ (A) and 6⁺ (B) charge states sprayed from native (black trace) and denaturing (red trace) conditions.

Figure 2.

Covalent modification of [ubiquitin+6H]⁶⁺ ionized from native conditions with [sulfo-HOAt]⁻. (A) Product ion spectrum of the ion/ion reaction between [ubiquitin + 6H] ⁶⁺ and [sulfo-HOAt – H]⁻ prior to activation. ♦ refers to electrostatic attachment of the reagent and * refers to covalent modification. (B) ATD of the full scan (mass range of 100 to 500 m/z) corresponding to ion/ion reactions between [ubiquitin + 6H]⁶⁺ and [sulfo-HOAt – H]-revealing the mobility separation of covalently modified products generated with different extents of modification. (C) Mass spectrum resulting from CID of the ion/ion reaction product (corresponding to 72 – 83 ms in the ATD).

Figure 3.

(A) Post-ion/ion reaction IM spectrum and mass spectra from zero (B), one (C), two (D), and three (E) anion attachments. ♦ refers to electrostatic attachment of the reagent and * refers to covalent modification.

Figure 4.

X-ray structure of ubiquitin (1ubq). The blue residues (K29, R54) are labeled under native conditions and the red (K33, K48) and green residue (K27) are labeled only under denaturing conditions. The red residues are protonated under native conditions and the green residue is buried and participates in a salt bridge with D52 (black). K11 is black as it participates in a salt bridge but in not labeled under any conditions. The black line between K27 and D52 represents the salt bridge.