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

Abstract

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.

A Novel Proteomics-Based Strategy for the Investigation of Peptide Sequences in Extraterrestrial Samples

“…APM stereoselectivity has already been used with success to differentiate neuropeptides containing D-amino acids from those including only L-residues in tissue extracts 15 18 . Very recently, an APM-based protocol has also enabled to indicate L to D-amino acids isomerization in peptides of the central nervous system in animals 19 . In this work, we propose a variation of this approach with the aim to search for peptide sequences in meteorites.…”

Polymer Conjugates of Antimicrobial Peptides (AMPs) with d-Amino Acids (d-aa): State of the Art and Future Opportunities

“…In terms of bioactivities, peptides with D-aa can exert neuroexcitatory [14,15] and cardioexcitatory roles [16], but also opioid [17] and anti-hypertensive [18] activity, endocrine function [19], and AM roles [20]. Peptides with D-aa have been proposed for cancer therapy [21] as adhesive biomaterials [22], vaccine adjuvants [23], and as inhibitors of pathological amyloid fibrillization [24][25][26][27][28].…”

Analysis of Peptide Stereochemistry in Single Cells by Capillary Electrophoresis–Trapped Ion Mobility Spectrometry Mass Spectrometry

“…MS has been successfully used for single cell chemical profiling, , but because MS alone is not inherently stereospecific, it must be coupled with complementary techniques to analyze endogenous DAACPs. High sensitivity and stereoselective tandem MS (MS/MS)-based methods have been developed to distinguish peptide diastereomers, − but have only occasionally been useful for detecting DAACPs in bulk tissue extracts; ,− moreover, fragmentation-based techniques have seldom been applied for the relative quantification of DAACPs in individual cells . This is in part because differentiation and quantitation of peptide diastereomers by MS/MS is not straightforward, and relies on product ion ratios rather than a physical separation of diastereomers. ,, …”

“…More specifically, sampling techniques and CE–MS approaches adapted to work with individual cells were combined with TIMS and used to characterize isomerized peptides. , CE enabled us to separate the peptides from individual neurons with minimal sample dilution, and TIMS allowed us to accurately assign the native peptide stereochemistry. The combined approaches facilitated the detection and simultaneous stereochemical characterization of three known isomerized neuropeptides derived from the pleurin precursor (NP_001191654.1) in single neurons isolated from the Aplysia californica central nervous system (CNS) …”

“…Based on our prior work, we know that in the A. californica CNS, Plrn1 exists as two possible stereoisomers, l -Plrn1 and d 2-Plrn1 ( d 2 indicates a d -residue at the second residue from the N-terminus); whereas Plrn2 and Plrn3 each exist as three possible stereoisomers, l -Plrn2/Plrn3, d 2-Plrn2/Plrn3, or d 3-Plrn2/Plrn3 ( d 3 indicates a d -residue at the third residue from the N-terminus) (Table ).…”