Department of Chemistry, The University of Western Ontario, London, Ontario N6A 5B7, Canada.
Anal Chem. 2022 May 31;94(21):7713-7721. doi: 10.1021/acs.analchem.2c01355. Epub 2022 May 19.
The transfer of peptide ions from solution into the gas phase by electrospray ionization (ESI) is an integral component of mass spectrometry (MS)-based proteomics. The mechanisms whereby gaseous peptide ions are released from charged ESI nanodroplets remain unclear. This is in contrast to intact protein ESI, which has been the focus of detailed investigations using molecular dynamics (MD) simulations and other methods. Under acidic liquid chromatography/MS conditions, many peptides carry a solution charge of 3+ or 2+. Because of this pre-existing charge and their relatively small size, prevailing views suggest that peptides follow the ion evaporation mechanism (IEM). The IEM entails analyte ejection from ESI droplets, driven by electrostatic repulsion between the analyte and droplet. Surprisingly, recent peptide MD investigations reported a different behavior, that is, the release of peptide ions droplet evaporation to dryness which represents the hallmark of the charged residue mechanism (CRM). Here, we resolved this conundrum by performing MD simulations on a common model peptide (bradykinin) in Rayleigh-charged aqueous droplets. The primary focus was on pH 2 conditions (bradykinin solution charge = 3+), but we also verified that our MD strategy captured pH-dependent charge state shifts seen in ESI-MS experiments. In agreement with earlier simulations, we found that droplets with initial radii of 1.5-3 nm predominantly release peptide ions the CRM. In contrast, somewhat larger radii (4-5 nm) favor IEM behavior. It appears that these are the first MD data to unequivocally demonstrate the viability of peptide IEM events. Electrostatic arguments can account for the observed droplet size dependence. In summary, both CRM and IEM can be operative in peptide ESI-MS. The prevalence of one over the other mechanism depends on the droplet size distribution in the ESI plume.
电喷雾电离（ESI）将肽离子从溶液转移到气相是基于质谱（MS）的蛋白质组学的一个组成部分。从带电 ESI 纳米液滴中释放气态肽离子的机制仍不清楚。这与完整蛋白质的 ESI 形成鲜明对比，后者一直是使用分子动力学（MD）模拟和其他方法进行详细研究的重点。在酸性液相色谱/ MS 条件下，许多肽带有 3+或 2+的溶液电荷。由于这种预先存在的电荷和它们相对较小的尺寸，普遍的观点认为肽遵循离子蒸发机制（IEM）。IEM 涉及由分析物与液滴之间的静电排斥驱动的从 ESI 液滴中逐出分析物。令人惊讶的是，最近的肽 MD 研究报告了一种不同的行为，即肽离子的释放 液滴蒸发至干燥，这代表了带电残基机制（CRM）的标志。在这里，我们通过在瑞利带电水液滴中对常见模型肽（缓激肽）进行 MD 模拟来解决这个难题。主要重点是 pH 2 条件（缓激肽溶液电荷= 3+），但我们还验证了我们的 MD 策略捕获了在 ESI-MS 实验中观察到的 pH 依赖性电荷状态转变。与早期的模拟结果一致，我们发现初始半径为 1.5-3nm 的液滴主要通过 CRM 释放肽离子。相比之下，稍大的半径（4-5nm）则有利于 IEM 行为。这似乎是首次明确证明肽 IEM 事件可行性的 MD 数据。静电论证可以解释观察到的液滴尺寸依赖性。总之，CRM 和 IEM 都可以在肽 ESI-MS 中起作用。一种机制相对于另一种机制的优势取决于 ESI 羽流中的液滴尺寸分布。
