Differential Post-Translational Amino Acid Isomerization Found among Neuropeptides in Aplysia californica

D-Amino acid-containing peptides (DAACPs) make up a class of post-translationally modified peptides in animals that play important roles as cell-to-cell signaling molecules. Despite the functional importance of L- to D-residue isomerization, little is known about its prevalence, mostly due to difficulties associated with detecting differences in peptide stereochemistry. Prior efforts to discover DAACPs have been largely focused on pursuing peptides based on homology to known DAACPs or DAACP-encoding precursors. Here, we used a combination of enzymatic screening, mass spectrometry, and chromatographic analysis to identify novel DAACPs in the central nervous system (CNS) of Aplysia californica. We identified five new DAACPs from the pleurin precursor and three DAACPs from previously uncharacterized proteins. In addition, two peptides from the pleurin precursor, Plrn2 and Plrn3, exist as DAACPs with the D-residue found at position 2 or 3. These differentially modified forms of Plrn2 and Plrn3 are located in specific regions of the animal's CNS. Plrn2 and Plrn3 appear to be the first animal DAACPs in which the D-residue is found at more than one position, and this suggests that L- to D-residue isomerization may be a more variable/dynamic modification than previously thought. Overall, this study demonstrates the utility of nontargeted DAACP discovery approaches for identifying new DAACPs and demonstrates that isomerization is prevalent throughout the CNS of A. californica.