Charge Loss of Proteins in vacuo: Implications for Native Mass Spectrometry and Ion Mobility

Abstract Native ion mobility−mass spectrometry (IM−MS) is a powerful biophysical technique for characterizing protein structure. Because individual charge states often exhibit distinct structural dynamics, accurate IM-MS measurements require charge states to be examined independently. Although anecdotal reports among mass spectrometrists suggest that protein ions can lose charges during native IM-MS experiments, a thorough investigation of this phenomenon has been lacking. Here, we demonstrate that protein ions undergo charge loss inside the mass spectrometer as a direct consequence of gas-phase collisions, potentially leading to altered charge state distributions and ion mobility data. This effect was consistently observed across two instrument platforms (Synapt G2-S and Q Exactive UHMR), both ion polarities (positive and negative mode) and for three proteins (bovine serum albumin, myoglobin, and immunoglobulin G). Systematic investigations of instrumental parameters revealed that collision gas pressure has the strongest impact on the magnitude of charge loss, and that higher charge states are more affected than lower charge states. We further show that this process is largely driven by the dissociation of salt adducts and can hence be minimized through desalting in solution or inside the mass spectrometer, prior to the first m/z-analyzer (usually a quadrupole). For collision-induced unfolding experiments, significant differences were found between fingerprints acquired with and without quadrupole selection, although it remains unclear whether this phenomenon is directly related to charge loss. Overall, our study provides fundamental insights into collision-induced charge loss as a source of error in native IM-MS measurements and discusses the relevance of quadrupole m/z-selection for improving confidence in IM studies.