Enhancing Metabolomic Coverage in Positive Ionization Mode Using Dicationic Reagents by Infrared Matrix-Assisted...

Mass spectrometry imaging is a powerful tool to analyze a large number of metabolites with their spatial coordinates collected throughout the sample. However, the significant differences in ionization efficiency pose a big challenge to metabolomic mass spectrometry imaging. To solve the challenge and obtain a complete data profile, researchers typically perform experiments in both positive and negative ionization modes, which is time-consuming. In this work, we evaluated the use of the dicationic reagent, 1,5-pentanediyl-bis(1-butylpyrrolidinium) difluoride (abbreviated to [C₅(bpyr)₂]F₂) to detect a broad range of metabolites in the positive ionization mode by infrared matrix-assisted laser desorption electrospray ionization mass spectrometry imaging (IR-MALDESI MSI). [C₅(bpyr)₂]F₂ at 10 µM was doped in 50% MeOH/H₂O (v/v) electrospray solvent to form +1 charged adducted ions with anionic species (-1 charged) through post-electrospray ionization. This method was demonstrated with sectioned rat liver and hen ovary. A total of 73 deprotonated metabolites from rat liver tissue sections were successfully adducted with [C₅(bpyr)₂]²⁺ and putatively identified in the adducted positive ionization polarity, along with 164 positively charged metabolite ions commonly seen in positive ionization mode, which resulted in 44% increased molecular coverage. In addition, we were able to generate images of hen ovary sections showing their morphological features. Following-up tandem mass spectrometry (MS/MS) indicated that this dicationic reagent [C₅(bpyr)₂]²⁺ could form ionic bonds with the headgroup of glycerophospholipid ions. The addition of the dicationic reagent [C₅(bpyr)₂]²⁺ in the electrospray solvent provides a rapid and effective way to enhance the detection of metabolites in positive ionization mode.