Thermal denaturation of holomyoglobin (hMb) in solution (10 mM ammonium acetate at pH = 4.5, 6.8, and 9.0) was monitored by ion mobility spectrometry (IMS) and mass spectrometry (MS) techniques to characterize the stability and investigate structural changes involved in unfolding. We utilize two experimental approaches to induce thermal denaturation: a variable-temperature electrospray ionization (vT-ESI) source that heats the bulk solution in the ESI emitter, and a variable-power 10.6 μm CO2 laser that rapidly heats nanodroplets produced by ESI. These two approaches sample different time scales of the denaturation process; long time scales (seconds to minutes) where the system is at equilibrium using the vT-ESI approach and shorter time scales (μs) by rapid droplet heating in which the system is in a pre-equilibrium state. Increasing the solution temperature (from 28 to 95 °C in the vT-ESI experiments) shifts the charge state distribution from low charge states ([M + 7H]7+ to [M + 9H]9+) to more highly charged species. This is accompanied by loss of the heme group to yield the apomyoglobin (aMb) species, indicating that the protein has unfolded. Monitoring the formation of aMb and the shift in average charge states of aMb and hMb with solution temperature allows for relative quantitation of their individual stabilities, highlighting the stabilizing effects of heme binding. We compare the degree of unfolding induced by heating the bulk solution (using vT-ESI) to the laser droplet heating approach and find that the rapid nature of the laser heating approach allows for transient pre-equilibrium states to be sampled.

中文翻译：

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用 IMS-MS 了解肌红蛋白的热变性：多重稳定结构和被捕获的预平衡状态的证据。

通过离子迁移谱 (IMS) 和质谱 (MS) 技术监测溶液（pH = 4.5、6.8 和 9.0 的 10 mM 乙酸铵）中全肌红蛋白 (hMb) 的热变性，以表征稳定性并研究参与的结构变化展开。我们利用两种实验方法来诱导热变性：加热 ESI 发射器中的本体溶液的变温电喷雾电离 (vT-ESI) 源，以及快速加热 ESI 产生的纳米液滴的可变功率 10.6 μm CO2 激光器。这两种方法对变性过程的不同时间尺度进行采样；使用 vT-ESI 方法使系统处于平衡状态的长时间尺度（秒到分钟）和通过快速液滴加热的更短时间尺度（μs），其中系统处于预平衡状态。增加溶液温度（在 vT-ESI 实验中从 28 °C 到 95 °C）将电荷态分布从低电荷态（[M + 7H]7+ 到 [M + 9H]9+）转移到更高电荷的物质。这伴随着血红素基团的损失以产生脱辅酶红蛋白 (aMb) 种类，表明该蛋白质已展开。监测 aMb 的形成以及 aMb 和 hMb 的平均电荷态随溶液温度的变化，可以对它们各自的稳定性进行相对定量，突出血红素结合的稳定作用。我们将通过加热本体溶液（使用 vT-ESI）与激光液滴加热方法诱导的展开程度进行比较，发现激光加热方法的快速特性允许对瞬态预平衡状态进行采样。