Spatially Mapping Neuropeptide Isomers via MALDI Trapped Ion Mobility MS Imaging

Imaging endogenous peptides with the chiral selectivity of their component amino acids remains an unmet analytical challenge. d-amino acid–containing peptides (DAACPs), formed by post-translational isomerization of l- to d-amino acids, make up a functionally important class of neuropeptides whose spatial distribution remains poorly understood. Here, we introduce untargeted imaging of neuropeptide stereoisomers using matrix-assisted laser desorption/ionization–trapped ion mobility mass spectrometry imaging (MALDI-TIMS-MSI). We mapped neuropeptides and their DAACP forms in the central nervous system (CNS) of Aplysia californica at single-cell resolution. We found that while both stereoisomeric forms of known neuropeptides were colocalized in nerves and neuropil, DAACPs were not detected in the neuronal soma. For example, the l-form of small cardioactive peptide B was detected at high levels in the B1 and B2 neurons of the buccal ganglion but the DAA-containing form was not detected; however, the DAACP form of small cardioactive peptide B was found in the neuropil of several ganglia. To confirm our assignments and eliminate isobaric interferences, we performed tandem MS with MALDI-TIMS-MSI. In total, we resolved 13 peptide stereoisomers from 6 endogenous neuropeptides. These results demonstrate that MALDI-TIMS-MSI is an effective approach for characterizing and mapping peptide stereoisomers in situ, providing critical insight into the spatial regulation of neuropeptide isomerization.