Mass Spectrometry Engineering Technology Research Center, Center for Advanced Measurement Science , National Institute of Metrology , Beijing 100029 , People's Republic of China.
Anal Chem. 2020 Jan 21;92(2):1770-1779. doi: 10.1021/acs.analchem.9b03416. Epub 2019 Dec 10.
Regulation of protein's charge state in electrospray is of great importance to the analysis of proteins. Different methods have been developed so far to increase the charge state of proteins. In this work, we investigated the influence of different anions on the charge state of proteins. Both strong acid anions and weak acid anions were taken into consideration. The results showed that the presence of 5 mM strong acid anions in acidic solutions could significantly increase the charge state of proteins. In comparison, weak acid anions with the same concentration in solution had little impact on the charge state of proteins. The species of the cations in the samples had very limited influence on the charge state. The presence of a certain amount of acid in sample solution was critical to the effect of strong acid anions. Almost no increase of the charge state was observed when no acid was added to the samples. However, remarkable increase of the charge state of myoglobin (Mb) was observed when 0.001% (v/v) acetic acid (HAc) was added to the sample together with 5 mM sodium chloride (NaCl). A higher concentration of acid in samples would further enhance the effect of strong acid anions on the increase of the charge state. Further investigations into the mechanism revealed that the effect of the strong acid anions on the charge state of proteins was based on the unfolding of the protein molecules during electrospray ionization (ESI). The interactions among H, anions, and protein molecules were so strong that it caused the unfolding of protein molecules and resulted in the increasing of proteins' charge states. The key factor that made strong acid anions and weak acid anions different in the results was the hydrolysis of the weak acid anions in acidic solutions. The present work furthers our understanding about electrospray, as well as the regulation of protein charge state. The presence of strong acid anions in acidic solutions can significantly influence the charge state of proteins in electrospray. Attention should be paid to this when regulating the charge state of proteins.
蛋白质在电喷雾中的荷质比的调控对于蛋白质分析非常重要。目前已经开发了多种方法来提高蛋白质的荷质比。在这项工作中，我们研究了不同阴离子对蛋白质荷质比的影响。考虑了强酸阴离子和弱酸阴离子。结果表明，在酸性溶液中存在 5 mM 的强酸阴离子可以显著增加蛋白质的荷质比。相比之下，相同浓度的弱酸阴离子对蛋白质的荷质比几乎没有影响。样品中阳离子的种类对荷质比的影响非常有限。样品溶液中存在一定量的酸对于强酸阴离子的效果是至关重要的。当样品中不加酸时，几乎观察不到荷质比的增加。然而，当向样品中加入 0.001%（v/v）的乙酸（HAc）以及 5 mM 的氯化钠（NaCl）时，肌红蛋白（Mb）的荷质比显著增加。样品中酸的浓度越高，强酸阴离子对荷质比增加的效果就越明显。进一步的机制研究表明，强酸阴离子对蛋白质荷质比的影响是基于蛋白质分子在电喷雾电离（ESI）过程中的展开。H、阴离子和蛋白质分子之间的相互作用非常强烈，导致蛋白质分子展开，从而增加蛋白质的荷质比。强酸阴离子和弱酸阴离子在结果上存在差异的关键因素是弱酸阴离子在酸性溶液中的水解。本工作进一步加深了我们对电喷雾以及蛋白质荷质比调控的理解。在酸性溶液中存在强酸阴离子可以显著影响蛋白质在电喷雾中的荷质比。在调控蛋白质荷质比时应注意这一点。
