Imidazolium Compounds as Internal Exchange Reporters for Hydrogen/Deuterium Exchange by Mass Spectrometry

Hydrogen–deuterium
exchange mass spectrometry (HDX-MS) is
a powerful tool for protein structure analysis that is well suited
for biotherapeutic development and characterization. Because HDX is
strongly dependent on solution conditions, even small variations in
temperature or pH can have a pronounced effect on the observed kinetics
that can manifest in significant run-to-run variability and compromise
reproducibility. Recent attention has been given to the development
of internal exchange reporters (IERs), which directly monitor changes
to exchange reaction conditions. However, the currently available
small peptide IERs are only capable of sampling a very narrow temporal
window and are understood to exhibit complex solution dependent exchange
behavior. Here we demonstrate the use of imidazolium carbon acids
as superior IERs for HDX-MS. These compounds exhibit predictable exchange
behavior under a wide variety of reaction conditions, are highly stable,
and can be readily modified to exchange over a broad temporal window.
The use of these compounds as IERs for solution based HDX-MS could
considerably extend the utility of the technique by allowing for more
robust empirical exchange correction, thereby improving reproducibility.

氢-氘交换质谱法（HDX-MS）是一种强大的蛋白质结构分析方法，其非常适合生物治疗药物的开发与表征。鉴于氢-氘交换过程对溶液条件高度敏感，即便温度或pH值的小幅变化也可能对观察到的动力学产生显著影响，从而导致实验结果间的显著差异，并损害重现性。近期，研究人员开始关注内部交换报告分子（IERs）的开发，这些分子能够直接监测交换反应条件的改变。然而，目前可用的短肽类IERs仅能采样极窄的时间窗口，并且被认为表现出复杂的溶液依赖性交换行为。本研究中，我们展示了咪唑碳酸类化合物作为HDX-MS优异的IERs的应用。这些化合物在广泛的反应条件下表现出可预测的交换行为，具有高度的稳定性，并且可以轻易地进行结构修饰以实现较宽时间窗口的交换。将这些化合物作为溶液基HDX-MS的IERs使用，将显著扩展该技术的应用范围，通过允许进行更稳健的经验性交换校正，从而提高实验的重现性。