Native Ion Mobility Mass Spectrometry: When Gas-Phase Ion Structures Depend on the Electrospray Charging Process

Abstract

Ion mobility spectrometry (IMS) has become popular to characterize biomolecule folding.Numerous studies have shown that proteins that are folded in solution remain folded in the gas phase, whereas proteins that are unfolded in solution adopt more extended conformations in the gas phase. Here, we discuss how general this tenet is. We studied single-stranded DNAs (human telomeric cytosine-rich sequences with CCCTAA repeats), which fold into an intercalated motif (i-motif) structure in a pH-dependent manner, thanks to the formation of C-H + -C base pairs. As i-motif formation is favored at low ionic strength, we could investigate the ESI-IMS-MS behavior of i-motif structures at pH ~5.5 over a wide range of ammonium acetate concentrations (15 mM to 100 mM). The control experiments consisted of either the same sequence at pH ~7.5, wherein the sequence is unfolded, or sequence variants that cannot form i-motifs (CTCTAA repeats). The surprising results came from the control experiments. We found that the ionic strength of the solution had a greater effect on the compactness of the gas-phase structures than the solution folding state. This means that electrosprayed ions keep a memory of the charging process, which is influenced by the electrolyte concentration. We discuss these results in light of the analyte partitioning between the droplet interior and droplet surface, which in turn influences the probability of being ionized via a charged residue-type pathway or a chain extrusion-type pathway.

Sizing up DNA nanostructure assembly with native mass spectrometry and ion mobility

“…We wondered if these findings could be due to structural changes of the DNA happening in the gas phase of the mass spectrometer. The study of biomolecular structure in the solution and gas phase is an active field of research, and both experimental and computational approaches have recently made significant progress in our understanding of the explicit role of solvation, and the consequences of its removal during the electrospray process 49 , 50 . Structures such as the large diameter barrels can obviously fluctuate and are in fact expected to undergo compaction in the gas phase due to the absence of water and the desire to self-solvate.…”

Probing ligand and cation binding sites in G-quadruplex nucleic acids by mass spectrometry and electron photodetachment dissociation sequencing

“…The gasphase compaction at low charge states was discussed before for DNA single strands, 48 DNA and RNA duplexes, 49 and DNA i-motifs. 50 At charge state 7-, the nonspecific interactions of the nonfolded forms are mostly broken, whereas those of the G-quadruplex persist up to higher internal energies. Charge states 7-and higher can be obtained only with the addition of sulfolane.…”

Native Mass Spectrometry and Nucleic Acid G-Quadruplex Biophysics: Advancing Hand in Hand

“…However, for molecules in which backbones can rearrange in the gas phase (or in the final desolvation/declustering stages of the electrospray process) to make nonspecific interactions, how can one infer a solution conformation (or conformational ensemble) from a collision cross section distribution? This is where we reach the current limits of structural ion mobility spectrometry: control experiments hint at the preservation of some memory of the solution conformation, yet the conformation is affected by the measurement, for example, by the electrospray charging process, ion internal energy, and time spent in the gas phase. The charging process is critical for nucleic acid rearrangements, as the phosphate groups are positioned along the backbone, and partial neutralization provides protons that can form non-native hydrogen bonds and which could be mobile …”