Is Native Mass\nSpectrometry in Ammonium Acetate Really\nNative? Protein Stability Differences in Biochemically Relevant Salt\nSolutions

Ammonium acetate is widely used in\nnative mass spectrometry\nto\nprovide adequate ionic strength without adducting to protein ions,\nbut different ions can preferentially stabilize or destabilize the\nnative form of proteins in solution. The stability of bovine serum\nalbumin (BSA) was investigated in 50 mM solutions of a variety of\nsalts using electrospray emitters with submicron tips to desalt protein\nions. The charge-state distribution of BSA is narrow (+14 to +18)\nin ammonium acetate (AmmAc), whereas it is much broader (+13 to +42)\nin solutions containing sodium acetate (NaAc), ammonium chloride (AmmCl),\npotassium chloride (KCl), and sodium chloride (NaCl). The average\ncharge state and percent of unfolded protein increase in these respective\nsolutions, indicating greater extents of protein destabilization and\nconformational changes. In contrast, no high charge states of either\nbovine carbonic anhydrase II or IgG1 were formed in AmmAc or NaCl\ndespite their similar melting temperatures to BSA, indicating that\nthe presence of unfolded BSA in some of these solutions is not an\nartifact of the electrospray ionization process. The charge states\nformed from the nonvolatile salt solutions do not change significantly\nfor up to 7 min of electrospray, but higher charging occurs after\n10 min, consistent with solution acidification. Formation of unfolded\nBSA in NaAc but not in AmmAc indicates that the cation identity, not\nacidification, is responsible for structural differences in these\ntwo solutions. Temperature-dependent measurements show both increased\ncharging and aggregation at lower temperatures in NaCl:Tris than in\nAmmAc, consistent with lower protein stability in the former solution.\nThese results are consistent with the order of these ions in the Hofmeister\nseries and indicate that data on protein stability in AmmAc may not\nbe representative of solutions containing nonvolatile salts that are\ndirectly relevant to biology.