Effects of aprotic solvents on the stability of metal‐free superoxide dismutase probed by native electrospray ionization–ion mobility–mass spectrometry

Abstract

Considering that aprotic solvents are often used as cosolvents in investigating the interactions between small molecules and proteins, we assessed the effects of five aprotic solvents represented by dimethylformamide (DMF) on the structure stabilities of metal‐free SOD1 (apo‐SOD1) by native electrospray ionization–ion mobility–mass spectrometry (ESI‐IM‐MS). These aprotic solvents include DMF, 1,3‐dimethyl‐2‐imidazolidinone (DMI), dimethyl sulfoxide (DMSO), acetonitrile (ACN), and tetrahydrofuran (THF). Results indicated that DMI, DMSO, and DMF at low percentage concentration could reduce the average charge and the dimer dissociation of apo‐SOD1. By contrast, ACN and THF at low concentration have no similar effect. DMF was selected as a representative solvent to further investigate the detailed effects on the structure stability of apo‐SOD1 by using collision‐induced dissociation and unfolding. The results reveal that the addition of minimal DMF to an aqueous protein solution can protect against the unfolding and dissociation of dimer, even under destabilizing conditions (such as low pH or high cone voltage). When the different percentage concentrations of DMF were added, the average collision cross section of apo‐SOD1 showed that apo‐SOD1 became compacted when the DMF concentration increased from 0% to 1% and eventually started extending when increased from 1% to 20%. The results indicated that DMF has similar effects to DMSO in native mass spectrometry (MS) and it can also be used as a cosolvent besides DMSO in investigating the stabilities of proteins and the interactions between small molecules and proteins.

Screening apo‐SOD1 conformation stabilizers from natural flavanones using native ion mobility mass spectrometry and fluorescence spectroscopy methods

“…In the study, the interaction between four flavanones and apo-SOD1 In previous studies, the dominating dimer species of apo-SOD1 were observed through ESI-MS, and the observed charge states ranged from +11 to +13. 24,28 Apo-SOD1 mainly exists in the form of dimers under the "near-native" experimental conditions. There is a little monomeric apo-SOD1.…”

“…apo-SOD1 was prepared by demetallation protocol. 24,25 Briefly, 1 mL of SOD1 stock solution was dialyzed for 24 h in a 20mM NH 4 OAC buffer (pH 3.2) containing 5mM EDTA. After 24 h, the dialysis solution was then replaced with 20mM NH 4 OAC buffer (pH 3.2) without EDTA.…”

Stabilities of superoxide dismutase and metal‐free superoxide dismutase studied by electrospray ionization ion mobility mass spectrometry

“…and apo-SOD1 can be calculated by the corresponding Equations 1 13 and 2. I di and I mon represent the exported peak intensities of the dimeric and monomeric species from the corresponding regions of the ion mobility profiles, respectively.…”