Department of Chemistry, The University of Western Ontario , London, Ontario N6A 5B7, Canada.
Department of Biochemistry, The University of Western Ontario , London, Ontario N6A 5C1, Canada.
Anal Chem. 2016 May 17;88(10):5345-54. doi: 10.1021/acs.analchem.6b00650. Epub 2016 Apr 27.
Electrospray ionization (ESI) allows the production of intact gas-phase ions from proteins in solution. Nondenaturing solvent conditions usually culminate in low ESI charge states. However, many mass spectrometric applications benefit from protein ions that are more highly charged. One way to boost protein charge is the addition of supercharging agents (SCAs) such as sulfolane or m-nitrobenzyl alcohol (m-NBA) to the aqueous solution. The supercharging mechanism remains controversial. We use molecular dynamics (MD) simulations to examine how SCAs affect the behavior of ESI nanodroplets. Simulations were conducted on myoglobin in water, water/sulfolane, and water/m-NBA. Na(+) ions served as surrogate charge carriers instead of H(+). We focus on conditions where the protein initially adopts its native conformation. MD-generated charge states show remarkable agreement with experimental data. Droplet shrinkage is accompanied by Na(+) ejection, consistent with the ion evaporation model (IEM). The droplets segregate into an outer SCA shell and an aqueous core. This core harbors protein and Na(+). Unfavorable SCA solvation restricts Na(+) access to the droplet surface, thereby impeding IEM ejection. Rapid water loss causes SCA enrichment, ultimately forcing all remaining Na(+) to bind the protein. IEM ejection is no longer feasible after this point, such that the protein becomes supercharged by Na(+) trapping. SCA-free droplets produce lower charge states because the aqueous environment ensures a higher IEM efficiency. For all scenarios examined here, proteins are released via solvent evaporation to dryness, as envisioned by the charged residue model. Our data provide the first atomistic view of the supercharging mechanism.
电喷雾电离 (ESI) 允许从溶液中的蛋白质中产生完整的气相离子。非变性溶剂条件通常导致低 ESI 电荷状态。然而，许多质谱应用受益于具有更高电荷的蛋白质离子。一种提高蛋白质电荷的方法是向水溶液中添加超荷试剂 (SCA)，例如环丁砜或间硝基苄醇 (m-NBA)。超荷机理仍存在争议。我们使用分子动力学 (MD) 模拟来研究 SCA 如何影响 ESI 纳米液滴的行为。在水中、水/环丁砜和水/m-NBA 中对肌红蛋白进行了模拟。Na(+) 离子用作替代电荷载体，而不是 H(+)。我们关注的是蛋白质最初采用其天然构象的条件。MD 生成的电荷状态与实验数据非常吻合。液滴收缩伴随着 Na(+) 的释放，这与离子蒸发模型 (IEM) 一致。液滴分离成一个外部 SCA 壳和一个水核。这个核心包含蛋白质和 Na(+)。不利的 SCA 溶剂化限制了 Na(+) 进入液滴表面，从而阻碍了 IEM 的释放。快速的水分流失导致 SCA 富集，最终迫使所有剩余的 Na(+)与蛋白质结合。在这一点之后，IEM 释放不再可行，因此蛋白质通过 Na(+) 捕获而被超荷。没有 SCA 的液滴产生较低的电荷状态，因为水相环境确保了更高的 IEM 效率。对于这里检查的所有情况，蛋白质通过溶剂蒸发释放到干燥状态，正如带电残基模型所设想的那样。我们的数据提供了超荷机理的第一个原子观点。
