Konermann L, Douglas D J
Department of Chemistry, University of British Columbia, Vancouver, Canada. konerman@
J Am Soc Mass Spectrom. 1998 Dec;9(12):1248-54. doi: 10.1016/S1044-0305(98)00103-2.
Electrospray ionization (ESI) mass spectrometry (MS) in both the positive and negative ion mode has been used to study protein unfolding transitions of lysozyme, cytochrome c (cyt c), and ubiquitin in solution. As expected, ESI of unfolded lysozyme leads to the formation of substantially 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 different behavior: In these three cases protein unfolding only leads to marginal changes in the negative ion charge state distributions, whereas in the positive ion mode pronounced shifts to higher charge states are observed. This shows that ESI MS in the negative ion mode as a method for probing conformational changes of proteins in solution should be treated with caution. The data presented in this work provide further evidence that the conformation of a protein in solution not its charge state is the predominant factor for determining the ESI charge state distribution in the positive ion mode. Furthermore, these data support the hypothesis of a recent study (Konermann and Douglas, Biochemistry 1997, 36, 12,296-12,302) which suggested that ESI in the positive ion mode is not sensitive to changes in the secondary structure of proteins but only to changes in the tertiary structure.
电喷雾电离（ESI）质谱（MS）已被用于研究溶液中溶菌酶、细胞色素c（cyt c）和泛素的蛋白质解折叠转变，采用了正离子和负离子模式。正如预期的那样，在两种操作模式下，未折叠的溶菌酶的ESI导致形成的电荷态比紧密折叠的蛋白质高得多。令人惊讶的是，cyt c的酸诱导解折叠以及泛素的酸和碱诱导解折叠表现出不同的行为：在这三种情况下，蛋白质解折叠仅导致负离子电荷态分布的微小变化，而在正离子模式下观察到明显向更高电荷态的转变。这表明，作为探测溶液中蛋白质构象变化的一种方法，负离子模式下的ESI MS应谨慎对待。这项工作中呈现的数据进一步证明，溶液中蛋白质的构象而非其电荷态是决定正离子模式下ESI电荷态分布的主要因素。此外，这些数据支持了最近一项研究（Konermann和Douglas，《生物化学》1997年，36卷，12296 - 12302页）的假设，该研究表明正离子模式下的ESI对蛋白质二级结构的变化不敏感，而仅对三级结构的变化敏感。
