title: A d-Amino Acid-Containing Neuropeptide Discovery Funnel.
abstract: 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.
authors: Itamar
authors: I
authors: Livnat
authors: Hua-Chia
authors: HC
authors: Tai
authors: Erik T
authors: ET
authors: Jansson
authors: Lu
authors: L
authors: Bai
authors: Elena V
authors: EV
authors: Romanova
authors: State Key Laboratory of Pharmaceutical Biotechnology, Collaborative Innovation Center of Chemistry for Life Sciences, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, Advanced Institute for Life Sciences, School of Life Sciences, Nanjing University , Nanjing, Jiangsu 210046, China.
authors: Ting-Ting
authors: TT
authors: Chen
authors: State Key Laboratory of Pharmaceutical Biotechnology, Collaborative Innovation Center of Chemistry for Life Sciences, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, Advanced Institute for Life Sciences, School of Life Sciences, Nanjing University , Nanjing, Jiangsu 210046, China.
authors: Ke
authors: K
authors: Yu
authors: State Key Laboratory of Pharmaceutical Biotechnology, Collaborative Innovation Center of Chemistry for Life Sciences, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, Advanced Institute for Life Sciences, School of Life Sciences, Nanjing University , Nanjing, Jiangsu 210046, China.
authors: Song-An
authors: SA
authors: Chen
authors: State Key Laboratory of Pharmaceutical Biotechnology, Collaborative Innovation Center of Chemistry for Life Sciences, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, Advanced Institute for Life Sciences, School of Life Sciences, Nanjing University , Nanjing, Jiangsu 210046, China.
authors: Yan
authors: Y
authors: Zhang
authors: State Key Laboratory of Pharmaceutical Biotechnology, Collaborative Innovation Center of Chemistry for Life Sciences, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, Advanced Institute for Life Sciences, School of Life Sciences, Nanjing University , Nanjing, Jiangsu 210046, China.
authors: Zheng-Yang
authors: ZY
authors: Wang
authors: State Key Laboratory of Pharmaceutical Biotechnology, Collaborative Innovation Center of Chemistry for Life Sciences, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, Advanced Institute for Life Sciences, School of Life Sciences, Nanjing University , Nanjing, Jiangsu 210046, China.
authors: Dan-Dan
authors: DD
authors: Liu
authors: Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai , New York, New York 10029, United States.
authors: Klaudiusz R
authors: KR
authors: Weiss
authors: State Key Laboratory of Pharmaceutical Biotechnology, Collaborative Innovation Center of Chemistry for Life Sciences, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, Advanced Institute for Life Sciences, School of Life Sciences, Nanjing University , Nanjing, Jiangsu 210046, China.|Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai , New York, New York 10029, United States.
authors: Jian
authors: J
authors: Jing
authors: Jonathan V
authors: JV
authors: Sweedler
mesh_terms: D000596:Amino Acids / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D001048:Aplysia / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D018826:CD13 Antigens / Q000378:metabolism; D013058:Mass Spectrometry; D009474:Neurons / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D009479:Neuropeptides / Q000032:analysis* / Q000378:metabolism
chemical_list: D000596:Amino Acids; D009479:Neuropeptides; D018826:CD13 Antigens