A D-amino acid-containing neuropeptide discovery funnel

Itamar Livnat, Hua Chia Tai, Erik T. Jansson, Lu Bai, Elena V. Romanova, Ting Ting Chen, Ke Yu, Song An Chen, Yan Zhang, Zheng Yang Wang, Dan Dan Liu, Klaudiusz R. Weiss, Jian Jing, Jonathan V. Sweedler

Research output: Contribution to journalArticlepeer-review

68 Scopus citations


A receptor binding class of D-amino acid-containing peptides (DAACPs) is formed in animals from an enzymatically mediated post-translational modification of ribosomally translated all-L-amino acid peptides. Although this modification can be required for biological actions, detecting it is challenging because DAACPs have the same mass as their all-L-amino acid counterparts. We developed a suite of mass spectrometry (MS) protocols for the nontargeted discovery of DAACPs and validated their effectiveness using neurons from Aplysia californica. The approach involves the following three steps, with each confirming and refining the hits found in the prior step. The first step is screening for peptides resistant to digestion by aminopeptidase M. The second verifies the presence of a chiral amino acid via acid hydrolysis in deuterium chloride, labeling with Marfey's reagent, and liquid chromatography-mass spectrometry to determine the chirality of each amino acid. The third involves synthesizing the putative DAACPs and comparing them to the endogenous standards. Advantages of the method, the D-amino acid-containing neuropeptide discovery funnel, are that it is capable of detecting the D-form of any common chiral amino acid, and the first two steps do not require peptide standards. Using these protocols, we report that two peptides from the Aplysia achatin-like neuropeptide precursor exist as GdYFD and SdYADSKDEESNAALSDFA. Interestingly, GdYFD was bioactive in the Aplysia feeding and locomotor circuits but SdYADSKDEESNAALSDFA was not. The discovery funnel provides an effective means to characterize DAACPs in the nervous systems of animals in a nontargeted manner. (Figure Presented).

Original languageEnglish
Pages (from-to)11868-11876
Number of pages9
JournalAnalytical Chemistry
Issue number23
StatePublished - 6 Dec 2016


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