In summary, the CCSD and CIU data are compatible with the fact that i-motifs are preserved from the solution to the gas phase. Native supercharging was essential to generate higher charge densities at which the differences are clearer. Ions with charge densities of z = 9 or 10 per 33 phosphate groups (z/P = 0.27 or 0.30) were the most favorable: they are still produced via a charged residue electrospray mechanism and thus preserve the solution interactions in the gas phase, yet the expand upon unfolding at a voltage that reflects the number and strength of preserved intramolecular hydrogen bonds. Ions with z/P of 0.21 or 0.24 underwent unfolding, but the process starts from a more compact state wherein nonspecific hydrogen bonding also contributes (ions with z/P < 0.2 did not extend upon collisional activation).