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

Area of Science:

• Biophysical Chemistry
• Analytical Chemistry
• Molecular Biophysics

Background:

• Ion mobility spectrometry (IMS) is widely used to study biomolecule folding.
• Typically, folded proteins in solution maintain their structure in the gas phase, while unfolded proteins adopt extended conformations.
• The generalizability of this principle across different biomolecules, like DNA, requires further investigation.

Purpose of the Study:

• To investigate the gas-phase behavior of DNA i-motif structures using ESI-IMS-MS.
• To determine if solution folding state or ionic strength has a greater influence on gas-phase DNA conformations.
• To explore the memory effects during electrospray ionization related to electrolyte concentration.

Main Methods:

• Electrospray Ionization-Ion Mobility Spectrometry-Mass Spectrometry (ESI-IMS-MS) was employed.
• Human telomeric cytosine-rich DNA sequences (CCCTAA repeats) were studied at pH ~5.5 (i-motif formation) and pH ~7.5 (unfolded).
• Experiments were conducted across a range of ammonium acetate concentrations (15-100 mM) to assess ionic strength effects.

Main Results:

• Solution ionic strength had a more pronounced effect on the compactness of gas-phase DNA structures than the solution folding state.
• Control experiments with unfolded DNA or non-i-motif forming sequences confirmed the dominant role of ionic strength.
• Electrosprayed ions retain a memory of the charging process, influenced by electrolyte concentration during droplet formation.

Conclusions:

• The established tenet of biomolecule folding transfer from solution to gas phase may not universally apply, particularly for DNA i-motifs.
• Electrolyte concentration during electrospray significantly influences gas-phase ion structure, potentially overriding solution folding.
• Analyte partitioning between droplet interior and surface during electrospray affects ionization pathways and subsequent gas-phase conformations.