Abstract
<jats:p>Native mass spectrometry (nMS) faces multiple challenges when utilized for high-mass targets like protein complexes or viral capsids. An important part of the nativeness in nMS is the use of ammonium acetate buffer (AmAc) rather than non-volatile salts, which mimics cellular conditions and is MS compatible. However, due to several drawbacks of AmAc, including its lack of buffering at pH 7, different buffer compounds have been proposed. This is often combined with the aim to charge reduce (CR) the highly charged target ions for easier interpretation of the different charge states. Here, we specifically look at alternative buffers to ammonium acetate and how effective they are at charge reducing. Further, activated ion mobility methods were used to probe the nativelike protein structures, comparing the structures between AmAc and alternative, charge reducing buffer conditions. Lastly, this paper proposes additions to current electrospray ionization (ESI) theory, by including the mechanical insight gained from these extensive CR experiments and the role of volatility, gas-phase basicity and hydrodynamic radius in determining the charge state distribution.</jats:p>