Division of Bioanalytical Chemistry, AIMMS Amsterdam Institute of Molecular and Life Sciences, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands.
Centre for Analytical Sciences Amsterdam, 1098XH Amsterdam, The Netherlands.
Anal Chem. 2020 Mar 17;92(6):4292-4300. doi: 10.1021/acs.analchem.9b04961. Epub 2020 Mar 6.
Size-exclusion chromatography employing aqueous mobile phases with volatile salts at neutral pH combined with electrospray-ionization mass spectrometry (SEC-ESI-MS) is a useful tool to study proteins in their native state. However, whether the applied eluent conditions actually prevent protein-stationary phase interactions, and/or protein denaturation, often is not assessed. In this study, the effects of volatile mobile phase additives on SEC retention and ESI of proteins were thoroughly investigated. Myoglobin was used as the main model protein, and eluents of varying ionic strength and pH were applied. The degree of interaction between protein and stationary phase was evaluated by calculating the SEC distribution coefficient. Protein-ion charge state distributions obtained during offline and online native ESI-MS were used to monitor alterations in protein structure. Interestingly, most of the supposedly mild eluent compositions induced nonideal SEC behavior and/or protein unfolding. SEC experiments revealed that the nature, ionic strength, and pH of the eluent affected protein retention. Protein-stationary phase interactions were effectively avoided using ammonium acetate at ionic strengths above 0.1 M. Direct-infusion ESI-MS showed that the tested volatile eluent salts seem to follow the Hofmeister series: no denaturation was induced using ammonium acetate (kosmotropic), whereas ammonium formate and bicarbonate (both chaotropic) caused structural changes. Using a mobile phase of 0.2 M ammonium acetate (pH 6.9), several proteins (i.e., myoglobin, carbonic anhydrase, and cytochrome c) could be analyzed by SEC-ESI-MS using different column chemistries without compromising their native state. Overall, with SEC-ESI-MS, the effect of nonspecific interactions between protein and stationary phase on the protein structure can be studied, even revealing gradual structural differences along a peak.
采用中性 pH 值下含挥发性盐的水性流动相的排阻色谱结合电喷雾电离质谱（SEC-ESI-MS）是研究天然状态下蛋白质的有用工具。然而，应用的洗脱条件是否确实能防止蛋白质-固定相相互作用和/或蛋白质变性，通常并未评估。在这项研究中，深入研究了挥发性流动相添加剂对 SEC 保留和蛋白质 ESI 的影响。肌红蛋白被用作主要的模型蛋白，并应用了不同离子强度和 pH 值的洗脱液。通过计算 SEC 分配系数来评估蛋白质与固定相的相互作用程度。在线和离线天然 ESI-MS 获得的蛋白质-离子电荷状态分布用于监测蛋白质结构的变化。有趣的是，大多数所谓的温和洗脱液组成会导致非理想的 SEC 行为和/或蛋白质展开。SEC 实验表明，洗脱液的性质、离子强度和 pH 值会影响蛋白质保留。使用离子强度高于 0.1 M 的乙酸铵可以有效地避免蛋白质与固定相的相互作用。直接进样 ESI-MS 表明，所测试的挥发性洗脱盐似乎遵循 Hofmeister 序列：使用乙酸铵（亲溶盐）不会引起变性，而甲酸铵和碳酸氢盐（均为疏溶盐）会引起结构变化。使用 0.2 M 乙酸铵（pH 6.9）作为流动相，使用不同的柱化学物质，可以通过 SEC-ESI-MS 分析几种蛋白质（如肌红蛋白、碳酸酐酶和细胞色素 c），而不会损害其天然状态。总的来说，通过 SEC-ESI-MS 可以研究蛋白质与固定相之间的非特异性相互作用对蛋白质结构的影响，甚至可以揭示沿着峰的逐渐结构差异。
