Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada, T6G 2G2.
J Am Soc Mass Spectrom. 2012 Dec;23(12):2122-31. doi: 10.1007/s13361-012-0483-y. Epub 2012 Sep 20.
Electrospray ionization mass spectrometry (ESI-MS) measurements were performed under a variety of solution conditions on a highly acidic sub-fragment (B3C) of the C-terminal carbohydrate-binding repeat region of Clostridium difficile toxin B, and two mutants (B4A and B4B) containing fewer acidic residues. ESI-MS measurements performed in negative ion mode on aqueous ammonium acetate solutions of B3C at low ionic strength (I < 80 mM) revealed evidence, based on the measured charge state distribution, of protein unfolding. In contrast, no evidence of unfolding was detected from ESI-MS measurements made in positive ion mode at low I or in either mode at higher I. The results of proton nuclear magnetic resonance and circular dichroism spectroscopy measurements and gel filtration chromatography performed on solutions of B3C under low and high I conditions suggest that the protein exists predominantly in a folded state in neutral aqueous solutions with I > 10 mM. The results of ESI-MS measurements performed on B3C in a series of solutions with high I at pH 5 to 9 rule out the possibility that the structural changes are related to ESI-induced changes in pH. It is proposed that unfolding of B3C, observed in negative mode for solutions with low I, occurs during the ESI process and arises due to Coulombic repulsion between the negatively charged residues and liquid/droplet surface charge. ESI-MS measurements performed in negative ion mode on B4A and B4B also reveal a shift to higher charge states at low I but the magnitude of the changes are smaller than observed for B3C.
电喷雾电离质谱（ESI-MS）测量在各种溶液条件下在艰难梭菌毒素 B 的 C 末端碳水化合物结合重复区的高度酸性亚片段（B3C）以及含有较少酸性残基的两个突变体（B4A 和 B4B）上进行。在低离子强度（I <80 mM）下，在水溶液中的 B3C 进行的负离子模式 ESI-MS 测量基于测量的电荷状态分布，显示出蛋白质展开的证据。相比之下，在低 I 或高 I 下的正离子模式下进行的 ESI-MS 测量未检测到展开的证据。在低 I 和高 I 条件下对 B3C 溶液进行的质子核磁共振和圆二色性光谱测量和凝胶过滤色谱结果表明，在 I > 10 mM 的中性水溶液中，该蛋白质主要以折叠状态存在。在一系列高 I（pH 5 至 9）溶液中对 B3C 进行的 ESI-MS 测量排除了结构变化与 ESI 诱导的 pH 变化有关的可能性。据推测，在低 I 溶液的负离子模式下观察到的 B3C 展开发生在 ESI 过程中，这是由于带负电荷的残基与液体/液滴表面电荷之间的库仑排斥引起的。在低 I 时，对 B4A 和 B4B 进行的负离子模式 ESI-MS 测量也揭示了电荷状态向更高电荷状态的转移，但变化幅度小于 B3C 观察到的变化幅度。
