Mass Spectrometry of Single GABAergic Somatic Motorneurons Identifies a Novel Inhibitory Peptide, As-NLP-22, in the Nematode Ascaris suum

• Published in: Journal of the American Society for Mass Spectrometry 2015-12, Vol.26 (12), p.2009-2023
• Main Authors: Konop, Christopher J., Knickelbine, Jennifer J., Sygulla, Molly S., Wruck, Colin D., Vestling, Martha M., Stretton, Antony O. W.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical Chemistry; Animals; Ascaris suum - chemistry; Ascaris suum - cytology; Ascaris suum - genetics; Ascaris suum - physiology; Base Sequence; Bioinformatics; Biotechnology; Chemistry; Chemistry and Materials Science; Cloning, Molecular; Desorption; Dissection; Focus: Mass Spectrometry-Based Strategies for Neuroproteomics and Peptidomics; Focus: Mass Spectrometry-Based Strategies for Neuroproteomics and Peptidomics: Research Article; GABAergic Neurons - chemistry; GABAergic Neurons - cytology; GABAergic Neurons - metabolism; Ionization; Ions; Lasers; Mass spectrometry; Molecular Sequence Data; Motor Neurons - chemistry; Motor Neurons - cytology; Motor Neurons - metabolism; Neuropeptides - analysis; Neuropeptides - genetics; Neuropeptides - metabolism; Organic Chemistry; Paralysis; Peptides; Proteomics; Scientific imaging; Sequence Alignment; Single-Cell Analysis - methods; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Spectroscopy; Synapses
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1007/s13361-015-1177-z

Neuromodulators have become an increasingly important component of functional circuits, dramatically changing the properties of both neurons and synapses to affect behavior. To explore the role of neuropeptides in Ascaris suum behavior, we devised an improved method for cleanly dissecting single motorneuronal cell bodies from the many other cell processes and hypodermal tissue in the ventral nerve cord. We determined their peptide content using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). The reduced complexity of the peptide mixture greatly aided the detection of peptides; peptide levels were sufficient to permit sequencing by tandem MS from single cells. Inhibitory motorneurons, known to be GABAergic, contain a novel neuropeptide, As-NLP-22 (SLASGRWGLRPamide). From this sequence and information from the A. suum expressed sequence tag (EST) database, we cloned the transcript ( As-nlp-22 ) and synthesized a riboprobe for in situ hybridization, which labeled the inhibitory motorneurons; this validates the integrity of the dissection method, showing that the peptides detected originate from the cells themselves and not from adhering processes from other cells (e.g., synaptic terminals). Synthetic As-NLP-22 has potent inhibitory activity on acetylcholine-induced muscle contraction as well as on basal muscle tone. Both of these effects are dose-dependent: the inhibitory effect on ACh contraction has an IC 50 of 8.3 × 10 –9 M. When injected into whole worms, As-NLP-22 produces a dose-dependent inhibition of locomotory movements and, at higher levels, complete paralysis. These experiments demonstrate the utility of MALDI TOF/TOF MS in identifying novel neuromodulators at the single-cell level. Graphical Abstract ᅟ