Investigating the Effect of Isoelectric Points on the Gas-Phase Stability of Native-like Proteins Analyzed in Positive- versus Negative-Ion Mode by IMS-MS

Native ion mobility spectrometry-mass spectrometry (IMS-MS) is routinely used for analysis of folded proteins and protein complexes. For many proteins, the three-dimensional structure is maintained during electrospray ionization (ESI) as the protein transitions to the gas phase, allowing for detailed investigation of the gaseous, ionic protein's structure and stability. Much of the native IMS-MS research has been conducted in positive-ion mode (+ESI), even when the protein of interest has a net-negative charge in solution at physiological pH. We hypothesize that analyzing a protein in the polarity that is opposite to its solution-phase charge, such as analyzing net-negative proteins by +ESI-MS, disrupts the network of noncovalent-bonding interactions within the protein to a greater extent than using the polarity that matches the protein's solution-phase charge, resulting in differences in protein stability. Herein, we show that while most protein ions have similar initial, folded structures in +ESI and negative-ion mode (-ESI), positive and negative ions exhibit significant differences in gas-phase stability. Furthermore, the energy required to cause this unfolding is often greater in the polarity corresponding to the solution-phase charge of the protein, indicating that the protein is more stable in that polarity. Thus, this work highlights the necessity of considering polarity when conducting native IMS-MS experiments.