Ozonization of Tissue Sections for MALDI MS Imaging of Carbon–Carbon Double Bond Positional Isomers of Phospholipids

Abstract

Visualizing the differential distribution of carbon–carbon double bond (CC db) positional isomers of unsaturated phospholipids (PL) in tissue sections by use of refined matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) technologies offers a high promise to deeper understand PL metabolism and isomer-specific functions in health and disease. Here we introduce an on-tissue ozonization protocol that enables a particular straightforward derivatization of unsaturated lipids in tissue sections. Collision-induced dissociation (CID) of MALDI-generated ozonide ions (with yields in the several ten percent range) produced the Criegee fragment ion pairs, which are indicative of CC db position(s). We used our technique for visualizing the differential distribution of Δ9 and Δ11 isomers of phosphatidylcholines in mouse brain and in human colon samples with the desorption laser spot size 15 μm, emphasizing the potential of the technique to expose local isomer-specific metabolism of PLs.

Identification of Phosphatidylcholine Isomers in Imaging Mass Spectrometry Using Gas-Phase Charge Inversion Ion/Ion Reactions

“…For example, tissue derivatization via Paternò-Büchi (PB) reactions has been used to identify double bond positions in lipids and tissue doping has been used to generate lithiated ion types to enable lipid acyl chain identification. [30][31][32][33][34][35] However, these methods can be time consuming, are irreversible, and can complicate the analysis due to off-target reactions and incomplete reactions. Several alternative ion activation strategies have been recently developed for use during imaging mass spectrometry experiments, including ion/molecule, ion/electron, and ion/photon reactions.…”

On‐tissue chemical derivatization in mass spectrometry imaging

“…Oxygen reacts with carbon db forming ozonide derivatives and reveals positional information through fragmentation. This has been introduced as an On-Tissue Chemical Derivatization in Mass Spectrometry Imaging | 17 on-tissue derivatization process investigating phosphatidylcholines (PCs) in mouse brain and in human colon samples (Bednařík et al, 2020). Ultraviolet photodissociation (UVPD) (193 nm) has been coupled with DESI to differentiate isomeric lipids from a variety of human tissue samples (Klein et al, 2018).…”

“…Addressing the same issue is the method of ozone-induced dissociation, which is based on the formation of ozonide derivatives when oxygen reacts with the lipid db, revealing positional information (Paine et al, 2018). Initially, this was developed to introduce ozone gas into the collision cell with the collision gas but has since been introduced as an on-tissue derivatization process investigating phosphatidylcholines (PCs) in mouse brain and in human colon samples (Bednařík et al, 2020).…”

Recent developments of novel matrices and on-tissue chemical derivatization reagents for MALDI-MSI

“…With this unique approach, specific lipid isomers were visualized in the rat brain. Later, Bednařík and coworkers [93] developed the approach for ozonization of unsaturated lipids under concentrated ozone atmosphere in a reaction chamber. The Criegee ozonide ions are formed after the addition of ozone to C=C double bond in PC 34:1 and the subsequent rearrangement.…”