Lou Xianwen, van Houtem Michel, Lafleur René P M, Schoenmakers Sandra M C, van Dongen Joost L J, Palmans Anja R A
Department of Chemical Engineering and Chemistry, Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, The Netherlands.
SyMO Chem B.V., Eindhoven, The Netherlands.
J Mass Spectrom. 2025 Jan;60(1):e5108. doi: 10.1002/jms.5108.
One critical issue in hydrogen/deuterium exchange mass spectrometry (HDX MS) analysis is the deleterious back exchange. Herein, we report that when matrix-assisted laser desorption/ionization (MALDI) is used, the MALDI process itself can also cause significant back exchange. The back exchange occurred inside the reactive MALDI plume was investigated by depositing a fully deuterated sample prepared in DO on top of a preloaded dried layer of matrix. A benzene-1,3,5-tricarboxamide (BTA) compound that can form supramolecular polymer in water and five peptides of angiotensin II (AT), pentaglycine (5G), pentaalanine (5A), cyclohexaglycine (C6G), and cyclohexaalanine (C6A) were selected as the testing compounds. Just like the situation in solution, the back exchange for the side chains and end groups is fast in the MALDI plume, while for the backbone amides, it is slow and dependent on the primary structure of the peptide. For the peptides tested, 5%-15% of D-labels in the backbone amides can be lost during the MALDI process. This degree of back exchange, although not an unbearable problem for most HDX MS applications as 85%-95% of the informative labels would still survive, could seriously limit the use of MALDI in the HDX MS analysis of supramolecular assemblies. For these assemblies, the EX1-like mechanism with two distinct distributions is common, and the back exchange could gravely distort or even merge the distinct isotopic distributions, which are the characteristic symbols of EX1.
氢/氘交换质谱（HDX MS）分析中的一个关键问题是有害的反向交换。在此，我们报告当使用基质辅助激光解吸/电离（MALDI）时，MALDI过程本身也会导致显著的反向交换。通过将在D₂O中制备的完全氘代样品沉积在预先加载的干燥基质层顶部，研究了反应性MALDI羽流内发生的反向交换。选择了一种能在水中形成超分子聚合物的苯-1,3,5-三甲酰胺（BTA）化合物以及血管紧张素II（AT）、五甘氨酸（5G）、五丙氨酸（5A）、环己六甘氨酸（C6G）和环己六甲氨酸（C6A）这五种肽作为测试化合物。与溶液中的情况一样，侧链和端基在MALDI羽流中的反向交换很快，而主链酰胺的反向交换则很慢且取决于肽的一级结构。对于所测试的肽，主链酰胺中5%-15%的D标记在MALDI过程中会丢失。这种程度的反向交换，虽然对于大多数HDX MS应用来说不是一个无法承受的问题，因为85%-95%的信息性标记仍然会保留，但可能会严重限制MALDI在超分子组装体的HDX MS分析中的使用。对于这些组装体，具有两种不同分布的类似EX1的机制很常见，并且反向交换可能会严重扭曲甚至合并不同的同位素分布，而这些分布是EX1的特征标志。
