Bothersome Back Exchange in MALDI Plume and Its Impact on Hydrogen/Deuterium Exchange Mass Spectrometry Analysis

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 D₂O 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.