Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) visualizes the distribution of phospho- and glycolipids in tissue sections. However, C=C double-bond (db) positional isomers generally cannot be distinguished. Now an on-tissue Paternò-Büchi (PB) derivatization procedure that exploits benzaldehyde as a MALDI-MSI-compatible reagent is introduced. Laser-induced postionization (MALDI-2) was used to boost the yields of protonated PB products. Collision-induced dissociation of these species generated characteristic ion pairs, indicative of C=C position, for numerous singly and polyunsaturated phospholipids and glycosphingolipids in mouse brain tissue. Several db-positional isomers of phosphatidylcholine and phosphatidylserine species were expressed with highly differential levels in the white and gray matter areas of cerebellum. Our PB-MALDI-MS/MS procedure could help to better understand the physiological role of these db-positional isomers.

Matrix‐assisted laser desorption ionization mass spectrometry imaging (MALDI‐MSI) visualizes the distribution of phospho‐ and glycolipids in tissue sections. However, C=C double‐bond (db) positional isomers generally cannot be distinguished. Now an on‐tissue Paternò–Büchi (PB) derivatization procedure that exploits benzaldehyde as a MALDI‐MSI‐compatible reagent is introduced. Laser‐induced postionization (MALDI‐2) was used to boost the yields of protonated PB products. Collision‐induced dissociation of these species generated characteristic ion pairs, indicative of C=C position, for numerous singly and polyunsaturated phospholipids and glycosphingolipids in mouse brain tissue. Several db‐positional isomers of phosphatidylcholine and phosphatidylserine species were expressed with highly differential levels in the white and gray matter areas of cerebellum. Our PB‐MALDI‐MS/MS procedure could help to better understand the physiological role of these db‐positional isomers.