The location and identity of phospholipids (PLs) within tissues can serve as diagnostic markers for tissue types or diseases. Whereas mass spectrometry imaging (MSI) has emerged as a powerful bioanalytical tool to visualize PL distributions, inferring PL identities from MSI experiments is challenging. Especially, CC double-bond (DB) positions are not identifiable in most MSI experiments. Herein, we introduce benzophenone (BPh) as a novel reactive matrix for matrix-assisted laser desorption/ionization (MALDI). BPh promotes desorption/ionization and simultaneously serves as derivatization reagent that allows functionalization of unsaturated PLs during the MALDI process via a laser-light driven Paternò–Büchi (PB) reaction without the need for additional equipment. Using BPh, PB product ions of numerous PL classes are readily generated to pinpoint the location of DBs. High lateral resolution MSI results of DB-position isomers are presented, highlighting the capabilities of BPh as a PB-reactive MALDI matrix to potentially unveil the impact of DB-position isomers in PL metabolism.

Local lipid variations in tissues are readily revealed with mass spectrometry imaging (MSI) methods, and the resulting lipid distributions serve as bioanalytical signatures to reveal cell-or tissue-specific lipids. Comprehensive MSI lipid mapping requires measurements in both ion polarities. Additionally, structural lipid characterization is necessary to link the lipid structure to lipid function. Whereas some structural elements of lipids are readily derived from high-resolution mass spectrometry (MS) and tandem-MS (MS n ), the localization of CC double bonds (DBs) requires specialized fragmentation and/or functionalization methods. In this work, we identify a multifunctional matrix-assisted laser desorption/ionization (MALDI) matrix for spatially resolved lipidomics investigations that reacts with lipids in Paterno−Buchi (PB) reactions during laser irradiation facilitating DB-position assignment and allows dual-polarity high-resolution MALDI-MSI and MALDI MS 2 I studies. By screening 12 compounds for improved ionization efficiency in positive-/negative-ion mode and the functionalization yield compared to the previously introduced reactive MALDI matrix benzophenone, 2-benzoylpyridine (BzPy) is identified as the best candidate. The new matrix enables DB localization of authentic standards belonging to 12 lipid classes and helps to assign 133/58 lipid features in positive-/negative-ion mode from mouse cerebellum tissue. The analytical capabilities of BzPy as a multifunctional MALDI-MSI matrix are demonstrated by imaging endogenous and PB-functionalized lipids in mouse kidney sections with 7 μm lateral resolution in both ion modes. Tracking diagnostic lipid DB-position fragment ions in mouse pancreatic tissue with down to 10 μm pixel size allows us to identify the islets of Langerhans associated with lipid isomer upregulation and depletion.

The effect of double bond functionalisation for selective double bond localisation by ultraviolet photodissociation of phosphatidylcholines is investigated. Paternò-Büchi reactions in nanoESI emitter tips enable attachment of acetophenone to double bonds of unsaturated phosphatidylcholines after 100 s of 254 nm light irradiation with about 50-80% reaction yield. Functionalized phosphatidylcholines dissociate upon 266 nm irradiation yielding double bond selective fragment ions in contrast to results for ultraviolet photodissociation of unmodified lipids. Ultraviolet photodissociation of Paternò-Büchi modified lipids results in a selectivity increase of up to 2.2 towards double bond localisation compared collision-induced dissociation experiments. Double bond localisation is also possible with ultraviolet photodissociation when alkali metal ion attachment to Paternò-Büchi modified phosphatidylcholines occurs in contrast to classic collision-induced dissociation experiments. The developed methodology is used to differentiate lipid double bond isomers and applied to phosphatidylcholines from egg yolk to identify 15 phosphatidylcholines. Results from this study demonstrate that locally depositing energy in close vicinity to cleavable bonds via ultraviolet photodissociation can result in increased dissociation selectivity. This method can help to disentangle contributions from different structural elements in complex tandem mass spectra of lipids and aid to the structural characterization of phospholipids in a "top-down" approach.

Local lipid variations in tissues are readily revealed with mass spectrometry imaging (MSI) methods and resulting lipid distributions serve as bioanalytical signatures to reveal cell- or tissue-specific lipids. Comprehensive MSI lipid mapping requires measurements in both ion polarities. Additionally, structural lipid characterization is necessary to link lipid structure to lipid function. Whereas some structural elements of lipids are readily derived from high-resolution mass spectrometry (MS) and tandem-MS (MSn), the localization of C=C double bonds (DBs) requires specialized fragmentation and/or functionalization methods. In this work, we identify a multifunctional matrix-assisted laser desorption/ionization (MALDI) matrix for spatially-resolved lipidomics investigations that reacts with lipids in Paternò-Büchi (PB) reactions during laser irradiation facilitating DB position assignment and allows dual polarity high-resolution MALDI-MSI and MALDI MS2I studies. By screening twelve compounds for improved ionization efficiency in positive/negative ion mode and PB functionalization yield compared to the previously introduced reactive MALDI matrix benzophenone, benzoylpyridine (BzPy) is identified as the best candidate. The multifunctional character of the new matrix enables DB localization of authentic standards belonging to twelve lipid classes and helps to assign 506/365 lipid features in positive/negative ion mode from mouse cerebellum tissue. The analytical capabilities of BzBy as a multifunctional MALDI-MSI matrix are demonstrated by imaging endogenous and PB-functionalized lipids in mouse kidney sections with 7 µm lateral resolution in both ion modes. Tracking diagnostic lipid DB position fragment ions in mouse pancreas tissue with down to 10 µm pixel size allows to identify islets of Langerhans associated lipid isomer upregulation or depletion. <br>