Azo-group reduction during the matrix-assisted laser desorption/ionization process in the presence of 2,5-dihydroxybenzoic acid

Some time ago, we published an announcement that the azo group that closes model cyclic peptides is often reduced in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) in the presence of 2,5-dihydroxybenzoic acid (2,5-DHB) as the matrix. In this work, we demonstrate that these peptides are ionized in all DHB matrix isomers, although threshold ionization laser energies as well as the reduction ratios differ in each matrix. Using a NALDI plate, we confirmed that their reduction depends on the presence of DHB matrix and that the hydrogen atoms participating in the reaction come from the DHB matrix hydroxyl group. We show that the reduction ratio is affected by the overall covalent structure of the peptide, by the presence of a free carboxyl group in DHB matrix, by the mutual position of the hydroxyl and carboxyl groups, as well as the laser beam intensity. Based on these results, it can be concluded that the azo-group reduction in cyclic peptides is a very complex process and we are far from fully understanding its nature. We hope that our experimental results will help to shed some light on the MALDI process that still remains mysterious in some of its aspects.