Journal
ACS CHEMICAL NEUROSCIENCE
Volume 1, Issue 7, Pages 505-519Publisher
AMER CHEMICAL SOC
DOI: 10.1021/cn1000217
Keywords
Neuropeptide; MALDI-TOF; nematode; Ascaris suum; single neuron; de nova sequencing
Funding
- National Institutes of Health [A115429, NRSA T32 GM007507]
- NCRR/SIG [S10RR024601]
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We have developed a method for dissecting single neurons from the nematode Ascaris suum, in order to determine their peptide content by mass spectrometry (MS). In this paper, we use MALDI-TOF MS and tandem MS to enumerate and sequence the peptides present in the two neurons, ALA and RID, that comprise the dorsal ganglion. We compare the peptide content determined by MS with the results of immunocytochemistry and in situ hybridization of previously isolated peptides AF2, AF8, and six peptides encoded by the afp-1 transcript. We find complete agreement among the three techniques, which validates single neuron MS as a method for peptide localization. We also discovered and sequenced six novel peptides in the ALA neuron. Cloning of cDNAs and database searching of Genomic Survey Sequences showed that transcript 0)42 encodes peptide AF36 (VPSAADM-MIRFamide) and afp-13 encodes AF19 (AEGLSSPLI-RFamide), AF34 (DSKLMDPLIRFamide), AF35 (DPQ-QRIVTDETVLRFamide), and three nonamidated peptides (PepTT, PepTL, and PepGE). We have found no similarities with reported peptide expression in the nematode Caenorhabditis elegans. This method promises to be ideally suited for determining the peptide content of each of the 298 neurons in the nervous system of this nematode.
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