3-Hydroxy-2-Nitrobenzoic Acid as a MALDI Matrix for In-Source Decay and Evaluation of the Isomers

In in-source decay (ISD) in matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS), 1,5-diaminonaphthalene (1,5-DAN) is a most frequently used matrix probably due to the highly sensitive detection of fragment ions. 1,5-DAN is a reducing matrix generating c- and z-series ions by N-Cα bond cleavage. However, it is difficult for reducing matrices to distinguish leucine and isoleucine, and generate c_(n-1)-series ions owing to proline (Pro) at residues n. Oxidizing matrices providing a- and x-series ions accompanied by d-series ions by Cα-C bond cleavage solve the problem, but their sensitivity of the ISD fragment ions has been lower than reducing matrices such as 1,5-DAN. Recently, 3-hydroxy-4-nitrobenzoic acid (3H4NBA) had been reported as an oxidizing matrix generating a-series ions with higher intensity compared with conventional oxidizing matrices such as 5-nitrosalicylic acid, but a little lower intensity compared with 1,5-DAN (Anal Chem 88, 8058-8063, 2016). In this study, 3H4NBA isomers (2H3NBA, 2H4NBA, 2H5NBA, 2H6NBA, 3H2NBA, 3H5NBA, 4H2NBA, 4H3NBA, 5H2NBA, and 3H4NBA) were evaluated. All the isomers generated a-series ions accompanied by d-series ions, wherein 3H2NBA, 3H5NBA, 4H2NBA, 4H3NBA, and 5H2NBA were first confirmed as oxidizing matrices for ISD. Among the isomers, 3H2NBA and 4H3NBA generated a-series ions with higher peak intensity compared with 3H4NBA for several peptides. Especially, 3H2NBA generated a-series ions with almost the same or higher intensity, and clearly higher peak resolution compared with c-series ions using 1,5-DAN in several cases. 3H2NBA was expected to contribute to ISD analyses in MALDI-MS as one of the most effective oxidizing matrices. Graphical Abstract ᅟ.