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 6 peptides encoded by the afp-1 transcript. We find complete agreement between the three techniques, which validates single neuron MS as a method for peptide localization. We also discovered and sequenced 6 novel peptides in the ALA neuron. Cloning of cDNAs and database searching of Genomic Survey Sequences showed that transcript afp-12 encodes peptide AF36 (VPSAADMMIRFamide), and afp-13 encodes AF19 (AEGLSSPLIRFamide), AF34 (DSKLMDPLIRFamide), AF35 (DPQQRIVTDETVLRFamide), and 3 non-amidated 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.

A tetrafluorophenyl (TFP) ester-terminated self-assembled monolayer (SAM) for the fabrication of DNA arrays on gold surfaces is described. Activated ester SAMs are desirable for biomolecule array fabrication because they readily react with amine-containing molecules to form a stable amide linkage. N-Hydroxysuccinimide (NHS) ester SAMs are commonly used for this purpose but are subject to a competing hydrolysis side reaction, limiting their effectiveness under basic conditions. TFP was evaluated here as an alternative activated ester leaving group with a potentially greater stability under basic conditions. It is shown that TFP SAMs are much more stable to basic pH than their NHS analogs and are also more hydrophobic, which is an advantage in the fabrication of high-density spotted arrays. DNA arrays prepared on TFP SAMs at pH 10 have a 5-fold greater surface density of DNA molecules, reduced fluorescence background, and smaller spot radii than those prepared on NHS SAM analogs.

We utilized three independent techniques, immunocytochemistry (ICC), single cell mass spectrometry (MS), and in situ hybridization (ISH), to localize neuropeptides and their transcripts in the nervous system of the nematode Ascaris suum . AF11 (SDIGISEPNFLRFa) is an endogenous peptide with potent paralytic effects on A. suum locomotory behavior. A highly specific antibody to AF11 showed robust immunostaining for AF11 in the paired AVK neurons in the ventral ganglion. We traced the processes from the AVK neurons into the ventral nerve cord and identified them as ventral cord interneurons. MS and MS/MS of single dissected AVKs detected AF11, two previously characterized peptides (AF25 and AF26), seven novel sequence-related peptides, including several sharing a PNFLRFamide C-terminus, and peptide NY, a peptide with an unrelated sequence. Also present in a subset of AVKs was AF2, a peptide encoded by the afp-4 transcript. By sequencing the afp-11 transcript, we discovered that it encodes AF11, all the AF11-related peptides detected by MS in AVK, and peptide NY. ISH detected the afp-11 transcript in AVK neurons, consistent with other techniques. ISH did not detect afp-11 in the ALA neuron, although both ICC and MS found AF11 in ca. 30% of ALAs. All 10 AF11-related peptides reduced acetylcholine-induced muscle contraction, but they differed in their rate of reversal of inhibition after removal of the peptide.

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to discover peptides in extracts of the large parasitic nematode Ascaris suum. This required the assembly of a new database of known and predicted peptides. In addition to those already sequenced, peptides were either previously predicted to be processed from precursor proteins identified in an A. suum library of expressed sequence tags (ESTs), or newly predicted from a library of A. suum genome survey sequences (GSSs). The predicted MS/MS fragmentation patterns of this collection of real and putative peptides were compared with the actual fragmentation patterns found in the MS/MS spectra of peptides fractionated by MS; this enabled individual peptides to be sequenced. Many previously identified peptides were found, and 21 novel peptides were discovered. Thus, this approach is very useful, despite the fact that the available GSS database is still preliminary, having only 1X coverage.

A monoclonal antibody, AF1-003, highly specific to the Ascaris suum neuropeptide AF1 (KNEFIRFamide), was generated. This antibody binds strongly to AF1 and extremely weakly to other peptides with C-terminal FIR-Famide: AF5 (SGKPTFIRFamide), AF6 (FIRFamide), and AF7 (AGPRFIRFamide). It does not recognize 35 other AF (A. suum FMRFamide-like) peptides at the highest concentration tested, nor does it recognize FMRFamide. When crude peptide extracts of A. suum are fractionated by two-step HPLC, the only fractions recognized by AF1-003 are those comigrating with synthetic AF1. By immunocytochemistry, antibody AF1-003 recognizes a small subset of the 298 neurons of A. suum: these include the paired URX and RIP neurons, two pairs of lateral ganglion neurons in the head, and the unpaired PQR and PDA or -B tail neurons that send processes to the head along the dorsal and ventral nerve cords, respectively. AF1 immunoreactivity is also seen in three pairs of pharyngeal neurons. Mass spectroscopy (MS) shows the presence of AF1 in the head, pharynx, and dorsal and ventral nerve cords. In A. suum, the neurons that contain AF1 show little overlap with neurons that express green fluorescent protein constructs targeting the flp-8 gene, which encodes AF1 in Caenorhabditis elegans (Kim and Li [2004] J. Comp. Neurol. 475:540– 550); the URX neurons express AF1 in both species, but, in C. elegans, flp-8 expression was not detected in RIP, PQR, and PDA or -B or in the pharynx. Other, less specific monoclonal antibodies recognize AF1, as well as other peptides to differing degrees; these antibodies are useful reagents for determination of neuronal morphology.