Distinguishing Endogenous d-Amino Acid-Containing Neuropeptides in Individual Neurons Using Tandem Mass Spectrometry

摘要

ABSTRACT: RNA-based protein synthesis produces L-amino acid-contain-ning proteins and peptides. D-amino acid-containing peptides (DAACPs) cannbe generated from L-amino acid peptides via post-translational modification.nIn the nervous system, the conformational change of a single L-amino acid inna peptide to its D-form results in altered bioactivity, with some DAACPsnhaving orders-of-magnitude enhanced efficacy. However, this modification isnoften overlooked when characterizing endogenous peptides. Here, with thenuse of matrix-assisted laser desorption ionization (MALDI) time-of-flightn(TOF)/TOF mass spectrometry, neuropeptides that have the secondnresidue isomerized to the D-isoform are distinguished from their L-epimersnvia differences in the relative amounts of specific fragment ions duringntandem MS. With the appropriate fragment ions chosen, and in some casesnwith the use of metal adducts, epimer discrimination is optimized. Specifi-ncally, the cardioexcitatory peptide Asn-DTrp-Phe-amide (NdWFa) was assayed directly from neurons isolated from the sea slugnAplysia californica; the fraction of the peptide with the second residue (W) in the D- versus L-form was 90 ( 10%. We demonstratenthat this approach is well suited for confirming DAACPs directly from cells and tissue, advancing our understanding of the L to Dnmodification and the role it plays in cell-to-cell signaling.