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. Many noncovalent interactions that dictate the protein’s native three-dimensional structure are 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 if 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 folded structures in +ESI and -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.