However, our data however revealed several potential pitfalls in the interpretation of CIU curves or unfolding voltages. First, the unfolding process can involve more ensembles than extracted with CIUsuite2, and the MCR-ALS extraction of pure components was key to extract hidden populations. Second, the sole fact of observing unfolding transitions does not indicate that an oligonucleotide was folded in solution (cfr. the CC control), only that it was folded in the gas phase, and this can stem from nonspecific hydrogen bonds forming in low charge states upon desolvation/declustering. The differences in collision voltages required for a particular transition to occur are relevant, and even here the choice of the control is key.