TY - JOUR
T1 - A D-amino acid-containing neuropeptide discovery funnel
AU - Livnat, Itamar
AU - Tai, Hua Chia
AU - Jansson, Erik T.
AU - Bai, Lu
AU - Romanova, Elena V.
AU - Chen, Ting Ting
AU - Yu, Ke
AU - Chen, Song An
AU - Zhang, Yan
AU - Wang, Zheng Yang
AU - Liu, Dan Dan
AU - Weiss, Klaudiusz R.
AU - Jing, Jian
AU - Sweedler, Jonathan V.
N1 - Publisher Copyright:
© 2016 American Chemical Society.
PY - 2016/12/6
Y1 - 2016/12/6
N2 - 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).
AB - 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).
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U2 - 10.1021/acs.analchem.6b03658
DO - 10.1021/acs.analchem.6b03658
M3 - Article
C2 - 27788334
AN - SCOPUS:85018912934
SN - 0003-2700
VL - 88
SP - 11868
EP - 11876
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 23
ER -