In contrast with proteins, nucleic acids electrosprayed in the negative mode from aqueous solutions at physiological ionic strength (150 mM NH4OAc) induce predominantly low charge states below the Rayleigh limit, independently of whether the strand was folded or not in solution. Ion mobility measurements and comparisons with calculated collision cross sections revealed that DNA as well as RNA single strands, duplex and hairpins present low charge states often more compact in the gas phase than in solution. Finally, upon collisional activation, ions at those charge states do not extend in the gas phase, but get more compact. Our problem is thus: how can we analyze nucleic acid structures from aqueous solutions at physiological ionic strength, produce ions that preserve the non-covalent interactions that were present in solution, yet with charge states high enough to undergo unfolding instead of compaction upon internal energy increase? Here we studied sequences containing i-motif structures, which are secondary structures mediated by hemi-protonated cytosine base pairs (see Figure ). The in vitro formation of i-motifs in solution has been demonstrated by different classical spectroscopic techniques, and their in vivo presence in human cells and control regulatory functions has been demonstrated. In the gas phase, intramolecular i-motif folding is hard to prove because the charge state represents the global protonation/deprotonation count, but does not tell whether protons are located in-between base pairs. Ion mobility or ion spectroscopy has to be used. The solution stability can be modulated by the sequence (e.g., a hairpin in the loop increases the stability of the entire structure, replacing cytosines by thymines disrupts the i-motif entirely)