Detecting D-amino acid-containing neuropeptides using selective enzymatic digestion

Neuropeptides, gene products that undergo extensive post-translational modification (PTM), are frequently characterized using mass spectrometry (MS). One PTM in particular, the conversion of an L-amino acid to a D-amino acid, has no associated mass shift. Therefore, this PTM is difficult to evaluate using MS alone, especially in complex peptide mixtures. Here, enzymatic digestion using microsomal alanyl aminopeptidase is combined with MS characterization. This enzyme selectively degrades peptides lacking a D-amino acid in the second position from the N-terminus. By comparing a sample before and after digestion, D-amino acid-containing peptides (DAACPs) present in small quantities in a complex mixture can be identified, even among much larger quantities of other non-DAACPs. Protocols that use microsomal alanyl aminopeptidase as a discovery-enabling agent are described and validated by identifying a known DAACP from the Aplysia californica abdominal ganglion.