Characterizing glycans is analytically challenging since glycans are heterogeneous, branched polymers with different three-dimensional conformations. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has been used to analyze native conformations and dynamics of biomolecules by measuring the mass increase of analytes as labile protons are replaced with deuterium following exposure to deuterated solvents. The rate of exchange is dependent on the chemical functional group, the presence of hydrogen bonds, pH, temperature, charge, and solvent accessibility. HDX-MS of carbohydrates is challenging due to the rapid exchange rate of hydroxyls. Here, we describe an observed HDX reaction associated with residual solvent vapors saturating electrospray sources. When undeuterated melezitose was infused after infusing D₂O, samples with up to 73% deuterium exchange were detected. This residual solvent HDX was observed for both carbohydrates and peptides in multiple instruments and dependent on sample infusion rate, infusion time, and deuterium content of the solvent. This residual solvent HDX was observed over several minutes of sample analysis and persisted long enough to alter the measured deuterium labeling and possibly change the interpretation of the results. This work illustrates that residual solvent HDX competes with in-solution HDX for rapidly exchanging functional groups. Thus, we propose conditions to minimize this effect, specifically for top-down, in-electrospray ionization, and quench-flow HDX experiments.

Open image in new window

由于聚糖是具有不同三维构象的异质，支链聚合物，因此表征聚糖在分析上具有挑战性。氢/氘交换质谱法（HDX-MS）已用于通过测量分析物的质量增加来分析生物分子的天然构象和动力学，因为不稳定的质子在暴露于氘代溶剂后被氘取代时，质子被氘取代。交换速率取决于化学官能团，氢键的存在，pH，温度，电荷和溶剂可及性。由于羟基的快速交换，碳水化合物的HDX-MS具有挑战性。在这里，我们描述了观察到的与残留溶剂蒸汽相关的HDX反应，这些溶剂蒸汽会使电喷雾源饱和。输注D ₂后注入未氘化的松三糖O，检测到氘交换率高达73％的样品。在多种仪器中都观察到了残留的溶剂HDX的碳水化合物和多肽，并取决于样品的注入速度，注入时间和溶剂的氘含量。在几分钟的样品分析中观察到了这种残留溶剂HDX，并持续了足够长的时间，以改变测得的氘标记，并可能改变结果的解释。这项工作说明残留溶剂HDX与溶液中HDX竞争快速交换官能团。因此，我们提出了将这种影响最小化的条件，特别是对于自顶向下，电喷雾中电离和急流HDX实验。

在新窗口中打开图像