Stevens Alexander, Abramsson Mia L, Agasid Mark T, Allison Timothy M, Landreh Michael
Department of Cell and Molecular Biology, Uppsala University, 751 24 Uppsala, Sweden.
Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, 171 65 Solna, Sweden.
Anal Chem. 2025 May 27;97(20):10738-10744. doi: 10.1021/acs.analchem.5c00757. Epub 2025 May 14.
Native mass spectrometry (nMS) is routinely used to monitor ligand binding in protein complexes. However, such interactions are easily distorted by unwanted dissociation during desolvation and ionization. This effect can be mitigated by chemical additives that modulate the ion charge, which results in lower-energy ions and better-preserved interactions. While positive-mode is the most used ionization approach, the use of negative-mode ionization has attracted attention due to its surprising ability to reduce the dissociation of some protein complexes. We therefore investigated whether charge-reducing compounds that stabilize proteins in positive ionization mode, such as imidazole, acetonitrile, and trimethylammonium--oxide, affect the charge and stability of complexes in negative ionization mode. Using the complex between myoglobin and its heme cofactor as a model system, we find that charge reduction efficiency is not maintained between positive and negative polarity, as expected for different charge reduction mechanisms. We also suggest that charge and stability correlate in positive and negative ion modes. Notably, the strongest stabilizing effects were observed for compounds that form electrospray adducts, which can dissociate during in-source activation and cool the desolvated ions. Together, the effects of charge-modulating additives suggest that relative ion stabilities may be directly comparable between polarities, although the protective effects of evaporative cooling have to be considered. We demonstrate the usefulness of the approach to detect cooperative stabilization of lysozyme by two epigallocatechin-3-gallate molecules, which requires negative ion mode and charge reduction. The strategic use of chemical additives in negative-mode ionization experiments may therefore be important for preserving complexes for analysis.
原生质谱（nMS）通常用于监测蛋白质复合物中的配体结合。然而，在去溶剂化和电离过程中，此类相互作用很容易因不必要的解离而扭曲。这种影响可以通过调节离子电荷的化学添加剂来减轻，这会产生能量较低的离子，并能更好地保留相互作用。虽然正模式是最常用的电离方法，但负模式电离因其在减少某些蛋白质复合物解离方面的惊人能力而受到关注。因此，我们研究了在正电离模式下稳定蛋白质的电荷减少化合物，如咪唑、乙腈和三甲基氧化铵，是否会影响负电离模式下复合物的电荷和稳定性。以肌红蛋白及其血红素辅因子之间的复合物作为模型系统，我们发现正负极性之间的电荷减少效率无法保持，这与不同的电荷减少机制预期一致。我们还表明，电荷与稳定性在正离子和负离子模式下存在关联。值得注意的是，对于形成电喷雾加合物的化合物观察到了最强的稳定作用，这些加合物在源内激活过程中可能会解离并冷却去溶剂化离子。总之，电荷调节添加剂的作用表明，尽管必须考虑蒸发冷却的保护作用，但相对离子稳定性在不同极性之间可能直接具有可比性。我们证明了该方法在检测两个表没食子儿茶素 - 3 - 没食子酸酯分子对溶菌酶的协同稳定作用方面的有用性，这需要负离子模式和电荷减少。因此，在负离子模式电离实验中战略性地使用化学添加剂对于保存复合物进行分析可能很重要。
