UNFOLDING OF PROTEINS MONITORED BY ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY - A COMPARISON OF POSITIVE AND NEGATIVE-ION MODES

Electrospray ionization (ESI) mass spectrometry (MS) in both the posit ive and negative ion mode has been used to study protein unfolding tra nsitions of lysozyme, cytochrome c (cyt c), and ubiquitin in solution. As expected, ESI of unfolded lysozyme leads to the formation of subst antially higher charge states than the tightly folded protein in both modes of operation. Surprisingly, the acid-induced unfolding of cyt c as well as the acid and the base-induced unfolding of ubiquitin show d ifferent behavior: In these three cases protein unfolding only leads t o marginal changes in the negative ion charge state distributions, whe reas in the positive ion mode pronounced shifts to higher charge state s are observed. This shows that ESI MS in the negative ion mode as a m ethod for probing conformational changes of proteins in solution shoul d be treated with caution. The data presented in this work provide fur ther evidence that the conformation of a protein in solution not its c harge state is the predominant factor for determining the ESI charge s late distribution in the positive ion mode. Furthermore, these data su pport the hypothesis of a recent study (Konermann and Douglas, Biochem istry 1997, 36, 12296-12302) which suggested that ESI in the positive ion mode is not sensitive to changes in the secondary structure of pro teins but only to changes in the tertiary structure. (C) 1998 American Society for Mass Spectrometry.