Separation of Isobaric Lipids in Imaging Mass Spectrometry Using Gas-Phase Charge Inversion Ion/Ion Reactions

The diverse array of chemical compounds present in tissue samples results in many isobaric (i.e., same nominal mass) compounds in imaging mass spectrometry experiments. Adequate separation and differentiation of these compounds is necessary to ensure accurate analyte identification and avoid composite images comprising multiple compounds. High-resolution accurate mass (HRAM) measurements are able to resolve these compounds in some instances, but HRAM measurements are not always feasible depending on the instrument platform and the desired experimental time scale. Alternatively, tandem mass spectrometry (MS/MS) can be used to perform gas-phase transformations that improve molecular specificity. While conventional MS/MS methods employ collision induced dissociation (CID) to fragment compounds of interest and then analyze the product masses, gas-phase ion/ion reactions can be used to instead selectively react with desired classes of analytes. Herein, we have used gas-phase charge inversion ion/ion reactions to selectively resolve phosphatidylcholines (PCs) in isobaric lipid mixtures. A 1,4-phenylenedipropionic acid (PDPA) reagent dianion readily reacts with [M + H]⁺, [M + Na]⁺, and [M + K]⁺ ion types to produce demethylated product anions for each PC, [PC - CH₃]^-. These product anions are no longer isobaric and now differ in mass by 22 Da (protonated versus sodiated) and 16 Da (sodiated versus potassiated), respectively. This reaction has been used to differentiate isobaric lipids in the imaging mass spectrometry analysis of rat brain tissue.