Abstract
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.
Supplementary materials
Title
Spatially Mapping Neuropeptides and Their Isomers by MALDI Trapped Ion Mobility MS Imaging
Description
Detailed method parameters for MALDI-TIMS imaging, a complete list of DAACPs analyzed in this study, mobility of peptide standards, on-tissue tandem MS spectra for Pleurin peptide isomers, regional mobility profile data, and LC-MS data for SCP peptides.
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