**5. Conclusions**

Single-molecule description of AS conformational ensemble in solution detects differently structured components that are overseen by bulk spectroscopic methods, which probe secondary structure, but are consistent with the different degrees of compactness suggested by CSD analysis. Thus, although ion-mobility studies and molecular-dynamics simulations have shown that IDPs rearrange in the gas phase in a charge-dependent fashion [40], the extent of ionization at the moment of transfer from solution to gas phase, i.e., CSDs, seems to reflect structural heterogeneity in solution rather than ESI artifacts. This correspondence is experimentally established here, independently of assumptions on the underlying ESI mechanism. Combined description by orthogonal biophysical methods can provide valuable constraints for computational simulations of IDP conformational ensembles in the presence or absence of interactors [51].

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/20/ 5181/s1.

**Author Contributions:** Conceptualization, R.G., V.C., D.S., and F.M.; methodology, R.C., V.C., C.A.M., A.N., and C.S.; software, R.C.; investigation, R.C., V.C., C.A.M., A.N., and C.S.; resources, R.G., F.M.; data curation, R.C., V.C., D.S., A.N., and C.S.; writing—original draft preparation, R.C., V.C., C.A.M., R.G., and F.M.; writing—review and editing, V.C., S.B., G.L., R.G., and F.M.; supervision, V.C., D.S., S.B., G.L., R.G., and F.M.

**Funding:** This research received no external funding.

**Acknowledgments:** We thank G. Cappelletti and J. Clarke for the kind gift of the DNA plasmids used in this work.

**Conflicts of Interest:** The authors declare no conflict of interest.
