Abstract
Collision-induced unfolding (CIU) has provided new levels of understanding of the stabilities and structure(s) for gas phase protein and protein complex ions formed by electrospray ionization (ESI). Variable-temperature (vT-ESI) data provide complementary information about temperature-induced folding/unfolding (TIU) reactions of solution phase ions. Results obtained by using CIU and TIU provide complementary information about stabilities of gas phase versus solution phase ions. Such comparisons may provide the most direct experimental approach to answer a long-standing question from Fred McLafferty: "For how long, under what conditions, and to what extent, can solution structure be retained without solvent?" Answers to this question require greater understanding of the (i) structure(s), stabilities, and dynamics of proteins/protein complexes in solution prior to ESI; (ii) effects of water removal by droplet fission and "freeze-drying" by evaporation of water from the nanodroplet; and (iii) potential reactions and structural changes that may occur as the ions traverse the heated capillary, the final stage in the transition to solvent-free gas phase ions. Here, we employ vT-ESI coupled with ion mobility-mass spectrometry (IM-MS) as a means to provide more detailed answers to the above-mentioned questions. Apo- and metalated-metallothionein-2A (MT), a cysteine-rich metal binding protein, and various proteoforms of transthyretin (TTR), a homotetrameric (56 kDa) retinol and thyroxine transporter protein complex were studied to examine distinct features of CIU and TIU across two different types of protein complexes. The results in this work shed light on the capabilities of CIU, TIU, and average charge state (Z(avg)) for probing the rugged energy landscape of native proteins and highlights the effects of water and cofactors (metal ions) on the structure and stabilities of proteins and protein complexes.