Spatially Mapping Neuropeptides and Their Isomers by MALDI Trapped Ion Mobility MS Imaging

Imaging endogenous peptides with chiral selectivity remains an unmet analytical challenge. D-amino acid-containing peptides (DAACPs), formed by post-translational isomerization of L- to D-amino acids, are 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 of A. 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 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 the B1 and B2 neurons in other ganglia. To confirm our assignments and eliminate isobaric interferences, we performed tandem MS with MALDI-TIMS-MSI. In total, we resolved thirteen peptide stereoisomers from six 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.