Manipulation of Gas-Phase Charge Inversion Ion/Ion Reaction Kinetics for Improved Phospholipid Identification in Imaging Mass Spectrometry

Phosphatidylcholines (PCs), which differ in fatty acyl chain lengths and degrees of unsaturation, often exist as complex mixtures of isomeric and isobaric compounds. Accurate structural identification of these lipids in imaging mass spectrometry (IMS) is essential for contextualizing their spatial distributions within tissue biochemistry. Gas-phase charge inversion ion/ion reactions offer a powerful approach to improve lipid identification by converting precursor ion types prior to dissociation, yielding more structurally informative fragmentation patterns. Herein, we employ a novel multiply charged reagent ion, 1,2,4,5-tetrakis(4-carboxylphenyl)benzene (TCPB), to perform charge inversion ion/ion reactions with protonated PC analytes. The use of higher reagent charge states improves the kinetics of the ion/ion reaction, reducing the time required for reactions to occur within an imaging experiment. Additionally, the use of higher reagent charge states results in more exothermic reactions, which facilitates consecutive fragmentation of ion/ion reaction complexes to the desired fatty acyl product ions without the need for supplemental activation, further improving the speed and efficiency of this process. This optimized workflow is applied in imaging mass spectrometry experiments to spatially map PC 34:1 isomers within rat brain tissue, revealing distinct spatial distributions for PC 16:0/18:1 and PC 18:1/16:0. These results underscore the importance of isomer resolution in lipid imaging and demonstrate the potential for exploiting reaction kinetics to improve ion/ion reaction isomer and isobar separation in imaging applications.