Inorganic salts and nonvolatile-buffer components affect the structure and stability of proteins, and some protein complexes are unable to maintain their function and structure without them. However, it is well-known that these components cause suppression of analyte ionization during the electrospray ionization process. Thus, to establish appropriate methods for observation of the intact ions of protein and DNA complexes by native mass spectrometry (native MS) in the presence of nonvolatile buffer components, we herein examined the effect of ammonium acetate addition to a model homotetramer protein, alcohol dehydrogenase (ADH), which was prepared in a range of nonvolatile buffers, including Tris-HCl, phosphate, and HEPES buffers. Furthermore, native MS of nucleosome core particle (NCP), a large protein-DNA complex, prepared in nonvolatile buffer, was also examined. Intact ADH and NCP ions could be observed upon the addition of ammonium acetate, but NCP does not require as high of a concentration of ammonium acetate as ADH. Well-resolved peaks with different charge numbers could be observed for NCP prepared in Tris-HCl by addition of a lower amount of ammonium acetate than for ADH. This suggests that the effects of additives on native MS of biomolecular complexes can vary depending on the intramolecular interactions present. More specifically, NCP is stabilized mainly by electrostatic interactions, whereas the ADH tetramer depends on the presence of hydrophobic interactions between the four subunits. The results presented herein therefore are expected to contribute to structural biology studies of unstable protein-DNA complexes that are formed transiently during the transcription process.
Keywords: native mass spectrometry; nonvolatile buffer; nucleosome core particle; protein complex; the additive effect.