Three Independent Techniques Localize Expression of Transcript <i>afp-11</i> and Its Bioactive Peptide Products to the Paired AVK Neurons in Ascaris suum: In Situ Hybridization, Immunocytochemistry, and Single Cell Mass Spectrometry

Jarecki, Jessica L.; Viola, India R.; Andersen, Kari M; Miller, Andrew H.; Ramaker, Megan A.; Vestling, Martha M.; Stretton, A. O. W.

doi:10.1021/cn3001334

Cited by 11 publications

(49 citation statements)

References 55 publications

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“…Furthermore, as immunochemistry allows the determination of neurite morphology facilitating neuronal identification, several antibodies highly specific to certain A. suum FLPs were recently generated (42, 76). Another approach for FLP localization consists of direct mass spectrometric analysis on dissected neuronal tissue without the need of an extensive extraction process (29, 76).…”

Section: Localization Of Flpsmentioning

confidence: 99%

The FMRFamide-Like Peptide Family in Nematodes

et al. 2014

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In the three decades since the FMRFamide peptide was isolated from the mollusk Macrocallista nimbosa, structurally similar peptides sharing a C-terminal RFamide motif have been identified across the animal kingdom. FMRFamide-like peptides (FLPs) represent the largest known family of neuropeptides in invertebrates. In the phylum Nematoda, at least 32 flp-genes are classified, making the FLP system of nematodes unusually complex. The diversity of the nematode FLP complement is most extensively mapped in Caenorhabditis elegans, where over 70 FLPs have been predicted. FLPs have shown to be expressed in the majority of the 302 C. elegans neurons including interneurons, sensory neurons, and motor neurons. The vast expression of FLPs is reflected in the broad functional repertoire of nematode FLP signaling, including neuroendocrine and neuromodulatory effects on locomotory activity, reproduction, feeding, and behavior. In contrast to the many identified nematode FLPs, only few peptides have been assigned a receptor and there is the need to clarify the pathway components and working mechanisms of the FLP signaling network. Here, we review the diversity, distribution, and functions of FLPs in nematodes.

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Section: Localization Of Flpsmentioning

confidence: 99%

The FMRFamide-Like Peptide Family in Nematodes

et al. 2014

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“…Synthetic peptide standards were not suitable for calibration of the instrument because the laser intensity needed for analysis of single cells was often far too high when analyzing pure standard, making it impossible to identify the monoisotopic peak of the ion needed for accurate calibration. Therefore, a reference spectrum from a freshly dissected A. suum AVK neuron, with known peptide peaks previously described by Jarecki et al (2012), 17 was used for external calibration of the instrument.…”

Section: Methodsmentioning

confidence: 99%

“…14, 18, 19 More recently, we have used mass spectrometry (MS) to detect and then to sequence neuropeptides. 12, 13, 17, 20–22 We have now extended this technique to the single cell level; mass spectrometric methods are sensitive enough both to detect and to sequence peptides from single dissected neurons. 17, 20 This approach is limited only by how successfully we can dissect single neurons.…”

Section: Introductionmentioning

confidence: 99%

“…12, 13, 17, 20–22 We have now extended this technique to the single cell level; mass spectrometric methods are sensitive enough both to detect and to sequence peptides from single dissected neurons. 17, 20 This approach is limited only by how successfully we can dissect single neurons. For some neurons, it is relatively easy, but for others, such as the ventral cord motorneurons, the close association between the neurons and hypodermis, in which most of the neurons are embedded, and the many other neural processes in the nerve cord, complicate the dissection.…”

Section: Introductionmentioning

confidence: 99%

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Different Neuropeptides Are Expressed in Different Functional Subsets of Cholinergic Excitatory Motorneurons in the Nematode Ascaris suum

Konop

Knickelbine

Sygulla

et al. 2015

ACS Chem. Neurosci.

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Neuropeptides are known to have dramatic effects on neurons and synapses; however, despite extensive studies of the motorneurons in the parasitic nematode Ascaris suum, their peptide content had not yet been described. We determined the peptide content of single excitatory motorneurons by mass spectrometry and tandem mass spectrometry. There are 2 subsets of ventral cord excitatory motorneurons, each with neuromuscular output either anterior or posterior to their cell body, mediating forward or backward locomotion, respectively. Strikingly, the two sets of neurons contain different neuropeptides, with AF9 and 6 novel peptides (As-NLP-21.1-6) in anterior projectors, and the 6 afp-1 peptides in addition to AF2 in posterior projectors. In situ hybridization confirmed the expression of these peptides, validating the integrity of the dissection technique. This work identifies new components of the functional behavioral circuit, as well as potential targets for anti-parasitic drug development.

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“…Detecting multiple predicted peptides by their molecular mass often is sufficient to assign peptides to a certain prohormone, in the same way as the peptide mass fingerprint method is widely used in proteomics for protein identification. This approach allows matching of the individual cell peptide profiles to electrophysiological activity [72], localization of molecular probes [73], or both [74, 75]. A unique two-step strategy hyphenates MS to targeted chemical analysis of immunocytochemical-selected peptidergic neurons containing selected biomarkers of interest [76].…”

Section: Choosing Your Peptidomics Modus Operandi: a Guide On Methodomentioning

confidence: 99%

Peptidomics for the discovery and characterization of neuropeptides and hormones

Romanova

Sweedler

2015

Trends in Pharmacological Sciences

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The discovery of neuropeptides as signaling molecules with paracrine or hormonal regulatory functions has led to trailblazing advances in physiology and fostered the characterization of numerous neuropeptide-binding G-protein coupled receptors (GPCRs) as potential drug targets. The impact on human health has been tremendous: approximately 30% of commercial drugs act via the GPCR pathway. However, about 25% of the GPCRs encoded by the mammalian genome still lack their pharmacological identity. Searching for the orphan GPCR endogenous ligands that likely are neuropeptides has proved to be a formidable task. Here we describe the mass spectrometry-based technologies and experimental strategies that have been successful in achieving high throughput characterization of endogenous peptides in nervous and endocrine systems. Keywordsbioinformatics; endogenous peptides; sequencing; mass spectrometry; nervous system; quantitation What is peptidomics?Genome and transcriptome sequencing is upon us, so why we are still looking for ways to identify bioactive peptides in living systems? Bold genomic research efforts have provided extraordinary insights into the inventory of GPCR-receptor genes, the most coveted targets in drug development [1]. Gene association and knockout studies have illuminated the roles of various peptide genes in pathological conditions and diseases in animal models [2,3]. Yet genetic investigations do not determine the actual peptide gene products, neuropeptides, and hormones that mediate vital body functions and complex behaviors. The immense challenge is due to the complexity of molecular readout; a single gene can produce many products as a result of single nucleotide polymorphisms, alternative gene splicing, post-translational processing of precursor proteins, and the addition of chemical post-translational Corresponding author: Sweedler, J.V., jsweedle@illinois.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript modifications (PTMs) of cleaved peptides that oftentimes cannot be inferred from genomic data. Even annotating peptide coding genes and in silico neuropeptidome prediction requires specialized expertise and bioinformatics tools [4][5][6].At the dawn of the new millennium, the term 'peptidomics' (see the workflow shown in Figure 1A) was formally adopted to describe a method for high throughput, direct measurement and structural characterization of the endogenous peptides present in a given biological sample (see detailed historical review by Schrader and co...

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Three Independent Techniques Localize Expression of Transcript afp-11 and Its Bioactive Peptide Products to the Paired AVK Neurons in Ascaris suum: In Situ Hybridization, Immunocytochemistry, and Single Cell Mass Spectrometry

Cited by 11 publications

References 55 publications

The FMRFamide-Like Peptide Family in Nematodes

The FMRFamide-Like Peptide Family in Nematodes

Different Neuropeptides Are Expressed in Different Functional Subsets of Cholinergic Excitatory Motorneurons in the Nematode Ascaris suum

Peptidomics for the discovery and characterization of neuropeptides and hormones

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