Spatially Mapping Neuropeptide Isomers via MALDI Trapped Ion Mobility MS Imaging

Area of Science:

• Neuroscience
• Analytical Chemistry
• Biochemistry

Background:

• D-amino acid-containing peptides (DAACPs) are functionally important neuropeptides, but their spatial distribution is poorly understood.
• Existing analytical methods struggle to image peptides based on the chirality of their amino acids.

Purpose of the Study:

• To develop and apply a novel untargeted imaging technique for neuropeptide stereoisomers.
• To map the spatial distribution of DAACPs and their l-amino acid counterparts in the central nervous system (CNS).

Main Methods:

• Utilized matrix-assisted laser desorption/ionization-trapped ion mobility mass spectrometry imaging (MALDI-TIMS-MSI) for untargeted imaging.
• Applied tandem mass spectrometry (MS/MS) for isomer confirmation and elimination of isobaric interferences.
• Analyzed the central nervous system of *Aplysia californica* at single-cell resolution.

Main Results:

• Successfully mapped neuropeptides and their DAACP forms in the CNS of *Aplysia californica* with single-cell resolution.
• Found that while both stereoisomeric forms were colocalized in nerves and neuropil, DAACPs were absent from neuronal soma.
• Resolved 13 peptide stereoisomers from 6 endogenous neuropeptides, including the small cardioactive peptide B.

Conclusions:

• MALDI-TIMS-MSI is a powerful tool for characterizing and mapping peptide stereoisomers *in situ*.
• Provides critical insights into the spatial regulation of neuropeptide isomerization within the nervous system.
• Advances the understanding of DAACP function and distribution in neuronal signaling.