Muscle Protein Synthesis in Response to Plant-Based Protein Isolates With and Without Added Leucine Versus Whey Protein in Young Men and Women.

Background: Plant-based protein supplements often contain lower amounts of leucine and other essential amino acids (EAAs), potentially making them less effective in stimulating muscle protein synthesis (MPS) than animal-based proteins. Combining plant proteins could improve the EAA profile and more effectively support MPS. Objectives: The aim of this study was to determine the effect of a novel plant-based blend protein (PBP), PBP with added leucine (PBP + Leu) to levels equivalent to whey protein isolate (WHEY) on aminoacidemia and MPS responses in young men and women. We hypothesized that PBP + Leu would stimulate MPS equivalent to WHEY, and both would be greater than PBP. Methods: We employed a randomized, double-blind, crossover study consisting of 3 separate study visits to compare PBP, PBP + Leu, and WHEY. To measure MPS response to ingestion of the supplements, a primed continuous infusion of L-[ring13C6] phenylalanine was administered for 8 h at each study visit. Skeletal muscle tissue and blood samples were collected to measure aminoacidemia and MPS. Results: All protein supplements increased mixed MPS above postabsorptive levels (P < 0.001). However, MPS increase following ingestion of PBP was less than that following ingestion of PBP + Leu (P = 0.002) and WHEY (P = 0.046). There were no differences in MPS between PBP + Leu and WHEY (P = 0.052). Conclusions: Consumption of PBP isolate with added leucine stimulated MPS to a similar extent as whey protein in young men and women. PBPs containing higher leucine content promote anabolism to a similar extent as animal-based proteins.This study was registered at clinicaltrials.gov as NCT05139160.

Coingestion of Collagen With Whey Protein Prevents Postexercise Decline in Plasma Glycine Availability in Recreationally Active Men.

Whey protein ingestion during recovery from exercise increases myofibrillar but not muscle connective protein synthesis rates. It has been speculated that whey protein does not provide sufficient glycine to maximize postexercise muscle connective protein synthesis rates. In the present study, we assessed the impact of coingesting different amounts of collagen with whey protein as a nutritional strategy to increase plasma glycine availability during recovery from exercise. In a randomized, double-blind, crossover design, 14 recreationally active men (age: 26 ± 5 years; body mass index: 23.8 ± 2.1 kg·m-2) ingested in total 30 g protein, provided as whey protein with 0 g (WHEY), 5 g (WC05); 10 g (WC10), and 15 g (WC15) of collagen protein immediately after a single bout of resistance exercise. Blood samples were collected frequently over 6 hr of postexercise recovery to assess postprandial plasma amino acid kinetics and availability. Protein ingestion strongly increased plasma amino acid concentrations (p < .001) with no differences in plasma total amino acid availability between treatments (p > .05). The postprandial rise in plasma leucine and essential amino acid availability was greater in WHEY compared with the WC10 and WC15 treatments (p < .05). Plasma glycine and nonessential amino acid concentrations declined following whey protein ingestion but increased following collagen coingestion (p < .05). Postprandial plasma glycine availability averaged -8.9 ± 5.8, 9.2 ± 3.7, 23.1 ± 6.5, and 39.8 ± 11.0 mmol·360 min/L in WHEY, WC05, WC10, and WC15, respectively (incremental area under curve values, p < .05). Coingestion of a small amount of collagen (5 g) with whey protein (25 g) is sufficient to prevent the decline in plasma glycine availability during recovery from lower body resistance-type exercise in recreationally active men.

The impact and utility of very low-calorie diets: the role of exercise and protein in preserving skeletal muscle mass.

PURPOSE OF REVIEW: Very low-calorie diets (VLCD) are used as a weight loss intervention, but concerns have been raised about their potential negative impact on lean mass. Here, we review the available evidence regarding the effects of VLCD on lean mass and explore their utility and strategies to mitigate reductions in skeletal muscle. RECENT FINDINGS: We observed that VLCD, despite their effects on lean mass, may be suitable in certain populations but have a risk in reducing lean mass. The extent of the reduction in lean mass may depend on various factors, such as the duration and degree of energy deficit of the diet, as well as the individual's starting weight and overall health. SUMMARY: VLCD may be a viable option in certain populations; however, priority needs to be given to resistance exercise training, and secondarily to adequate protein intake should be part of this dietary regime to mitigate losing muscle mass.

Antifungal Activity of a Medical-Grade Honey Formulation against Candida auris.

Candida auris is a pathogenic yeast causing outbreaks in intensive care units with high mortality rates. The treatment of C. auris colonization is challenging due to high resistance rates. A potential alternative antifungal treatment is medical-grade honey. In this study the susceptibility of C. auris and other Candida species to the medical-grade honey-based formulation L-Mesitran® Soft was investigated. The medical-grade honey formulation reduced the growth of C. auris and other Candida species in a dose-dependent manner. This inhibition was not only due to the honey component, as treatment with an identical concentration of this component only was less effective and even stimulated the growth of C. albicans and C. glabrata, supporting the interpretation that supplements in the medical-grade honey formulation enhanced the antimicrobial activity. Increasing the concentration of the honey component to 40%, as is also present in an undiluted medical-grade honey formulation, lead to a 1- to 4-log inhibition of all Candida species. Unprocessed local honey reduced the growth of nearly all Candida species more strongly than medical-grade honey. C. auris' susceptibility to the medical-grade honey formulation did not depend on geographic origin or resistance profile, although the multiresistant isolates tended to be more susceptible. Altogether, medical-grade honey formulation has a strong antifungal activity against C. auris and other Candida species. Future studies should demonstrate whether the treatment of open wounds or skin colonized with C. auris is feasible and effective in the clinical setting.

Characterization and pharmacological analysis of two adipokinetic hormone receptor variants of the tsetse fly, Glossina morsitans morsitans.

Adipokinetic hormones (AKH) are well known regulators of energy metabolism in insects. These neuropeptides are produced in the corpora cardiaca and perform their hormonal function by interacting with specific G protein-coupled receptors (GPCRs) at the cell membranes of target tissues, mainly the fat body. Here, we investigated the sequences, spatial and temporal distributions, and pharmacology of AKH neuropeptides and receptors in the tsetse fly, Glossina morsitans morsitans. The open reading frames of two splice variants of the Glomo-akh receptor (Glomo-akhr) gene and of the AKH neuropeptide encoding genes, gmmhrth and gmmakh, were cloned. Both tsetse AKHR isoforms show strong sequence conservation when compared to other insect AKHRs. Glomo-AKH prepropeptides also have the typical architecture of AKH precursors. In an in vitro Ca(2+) mobilization assay, Glomo-AKH neuropeptides activated each receptor isoform up to nanomolar concentrations. We identified structural features of tsetse AKH neuropeptides essential for receptor activation in vitro. Gene expression profiles suggest a function for AKH signaling in regulating Glossina energy metabolism, where AKH peptides are released from the corpora cardiaca and activate receptors mainly expressed in the fat body. This analysis of the ligand-receptor coupling, expression, and pharmacology of the two Glomo-AKHR variants facilitates further elucidation of the function of AKH in G. m. morsitans.

FRPR-4 Is a G-Protein Coupled Neuropeptide Receptor That Regulates Behavioral Quiescence and Posture in Caenorhabditis elegans.

Neuropeptides signal through G-protein coupled receptors (GPCRs) to regulate a broad array of animal behaviors and physiological processes. The Caenorhabditis elegans genome encodes approximately 100 predicted neuropeptide receptor GPCRs, but in vivo roles for only a few have been identified. We describe here a role for the GPCR FRPR-4 in the regulation of behavioral quiescence and locomotive posture. FRPR-4 is activated in cell culture by several neuropeptides with an amidated isoleucine-arginine-phenylalanine (IRF) motif or an amidated valine-arginine-phenylalanine (VRF) motif at their carboxy termini, including those encoded by the gene flp-13. Loss of frpr-4 function results in a minor feeding quiescence defect after heat-induced cellular stress. Overexpression of frpr-4 induces quiescence of locomotion and feeding as well as an exaggerated body bend posture. The exaggerated body bend posture requires the gene flp-13. While frpr-4 is expressed broadly, selective overexpression of frpr-4 in the proprioceptive DVA neurons results in exaggerated body bends that require flp-13 in the ALA neuron. Our results suggest that FLP-13 and other neuropeptides signal through FRPR-4 and other receptors to regulate locomotion posture and behavioral quiescence.

Characterization of G protein-coupled receptors by a fluorescence-based calcium mobilization assay.

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous Gα16 construct is co-transfected. Following ligand binding, activation of the Gα16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous Gα16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The essence of insect metamorphosis and aging: electrical rewiring of cells driven by the principles of juvenile hormone-dependent Ca(2+)-homeostasis.

In holometabolous insects the fall to zero of the titer of Juvenile Hormone ends its still poorly understood "status quo" mode of action in larvae. Concurrently it initiates metamorphosis of which the programmed cell death of all internal tissues that actively secrete proteins, such as the fat body, midgut, salivary glands, prothoracic glands, etc. is the most drastic aspect. These tissues have a very well developed rough endoplasmic reticulum, a known storage site of intracellular Ca(2+). A persistent high [Ca(2+)]i is toxic, lethal and causal to apoptosis. Metamorphosis becomes a logical phenomenon if analyzed from: (1) the causal link between calcium toxicity and apoptosis; (2) the largely overlooked fact that at least some isoforms of Ca(2+)-ATPases have a binding site for farnesol-like endogenous sesquiterpenoids (FRS). The Ca(2+)-ATPase blocker thapsigargin, like JH a sesquiterpenoid derivative, illustrates how absence of JH might work. The Ca(2+)-homeostasis system is concurrently extremely well conserved in evolution and highly variable, enabling tissue-, developmental-, and species specificity. As long as JH succeeds in keeping [Ca(2+)]i low by keeping the Ca(2+)-ATPases pumping, it acts as "the status quo" hormone. When it disappears, its various inhibitory effects are lifted. The electrical wiring system of cells, in particular in the regenerating tissues, is subject to change during metamorphosis. The possibility is discussed that in vertebrates an endogenous farnesol-like sesquiterpenoid, probably farnesol itself, acts as a functional, but hitherto completely overlooked Juvenile anti-aging "Inbrome", a novel concept in signaling.

Ancient neuromodulation by vasopressin/oxytocin-related peptides.

Neuropeptidergic signaling is widely adopted by animals for the regulation of physiology and behavior in a rapidly changing environment. The vasopressin/oxytocin neuropeptide family originates from an ancestral peptide precursor in the antecedent of protostomian and deuterostomian animals. In vertebrates, vasopressin and oxytocin have both hormonal effects on peripheral target tissues, such as in the regulation of reproduction and water balance, and neuromodulatory actions in the central nervous system controlling social behavior and cognition. The recent identification of vasopressin/oxytocin-related signaling in C. elegans reveals that this peptidergic system is widespread among nematodes. Genetic analysis of the C. elegans nematocin system denotes vasopressin/oxytocin-like peptides as ancient neuromodulators of neuronal circuits involved in reproductive behavior and associative learning, whereas former invertebrate studies focused on conserved peripheral actions of this peptide family. Nematocin provides neuromodulatory input into the gustatory plasticity circuit as well as into distinct male mating circuits to generate a coherent mating behavior. Molecular interactions are comparable to those underlying vasopressin- and oxytocin-mediated effects in the mammalian brain. Understanding how the vasopressin/oxytocin family fine-tunes neuronal circuits for social behavior, learning and memory poses a major challenge. Functional conservation of these effects in nematodes and most likely in other invertebrates enables the development of future models to help answering this question.

Neuropeptide secreted from a pacemaker activates neurons to control a rhythmic behavior.

BACKGROUND: Rhythmic behaviors are driven by endogenous biological clocks in pacemakers, which must reliably transmit timing information to target tissues that execute rhythmic outputs. During the defecation motor program in C. elegans, calcium oscillations in the pacemaker (intestine), which occur about every 50 s, trigger rhythmic enteric muscle contractions through downstream GABAergic neurons that innervate enteric muscles. However, the identity of the timing signal released by the pacemaker and the mechanism underlying the delivery of timing information to the GABAergic neurons are unknown. RESULTS: Here, we show that a neuropeptide-like protein (NLP-40) released by the pacemaker triggers a single rapid calcium transient in the GABAergic neurons during each defecation cycle. We find that mutants lacking nlp-40 have normal pacemaker function, but lack enteric muscle contractions. NLP-40 undergoes calcium-dependent release that is mediated by the calcium sensor, SNT-2/synaptotagmin. We identify AEX-2, the G-protein-coupled receptor on the GABAergic neurons, as the receptor for NLP-40. Functional calcium imaging reveals that NLP-40 and AEX-2/GPCR are both necessary for rhythmic activation of these neurons. Furthermore, acute application of synthetic NLP-40-derived peptide depolarizes the GABAergic neurons in vivo. CONCLUSIONS: Our results show that NLP-40 carries the timing information from the pacemaker via calcium-dependent release and delivers it to the GABAergic neurons by instructing their activation. Thus, we propose that rhythmic release of neuropeptides can deliver temporal information from pacemakers to downstream neurons to execute rhythmic behaviors.

Neuropeptide GPCRs in C. elegans.

Like most organisms, the nematode Caenorhabditis elegans relies heavily on neuropeptidergic signaling. This tiny animal represents a suitable model system to study neuropeptidergic signaling networks with single cell resolution due to the availability of powerful molecular and genetic tools. The availability of the worm's complete genome sequence allows researchers to browse through it, uncovering putative neuropeptides and their cognate G protein-coupled receptors (GPCRs). Many predictions have been made about the number of C. elegans neuropeptide GPCRs. In this review, we report the state of the art of both verified as well as predicted C. elegans neuropeptide GPCRs. The predicted neuropeptide GPCRs are incorporated into the receptor classification system based on their resemblance to orthologous GPCRs in insects and vertebrates. Appointing the natural ligand(s) to each predicted neuropeptide GPCR (receptor deorphanization) is a crucial step during characterization. The development of deorphanization strategies resulted in a significant increase in the knowledge of neuropeptidergic signaling in C. elegans. Complementary localization and functional studies demonstrate that neuropeptides and their GPCRs represent a rich potential source of behavioral variability in C. elegans. Here, we review all neuropeptidergic signaling pathways that so far have been functionally characterized in C. elegans.

Vasopressin/oxytocin-related signaling regulates gustatory associative learning in C. elegans.

Vasopressin- and oxytocin-related neuropeptides are key regulators of animal physiology, including water balance and reproduction. Although these neuropeptides also modulate social behavior and cognition in mammals, the mechanism for influencing behavioral plasticity and the evolutionary origin of these effects are not well understood. Here, we present a functional vasopressin- and oxytocin-like signaling system in the nematode Caenorhabditis elegans. Through activation of its receptor NTR-1, a vasopressin/oxytocin-related neuropeptide, designated nematocin, facilitates the experience-driven modulation of salt chemotaxis, a type of gustatory associative learning in C. elegans. Our study suggests that vasopressin and oxytocin neuropeptides have ancient roles in modulating sensory processing in neural circuits that underlie behavioral plasticity.

PDF receptor signaling in Caenorhabditis elegans modulates locomotion and egg-laying.

In Caenorhabditis elegans, pdfr-1 encodes three receptors of the secretin receptor family. These G protein-coupled receptors are activated by three neuropeptides, pigment dispersing factors 1a, 1b and 2, which are encoded by pdf-1 and pdf-2. We isolated a PDF receptor loss-of-function allele (lst34) by means of a mutagenesis screen and show that the PDF signaling system is involved in locomotion and egg-laying. We demonstrate that the pdfr-1 mutant phenocopies the defective locomotor behavior of the pdf-1 mutant and that pdf-1 and pdf-2 behave antagonistically. All three PDF receptor splice variants are involved in the regulation of locomotor behavior. Cell specific rescue experiments show that this pdf mediated behavior is regulated by neurons rather than body wall muscles. We also show that egg-laying patterns of pdf-1 and pdf-2 mutants are affected, but not those of pdfr-1 mutants, pointing to a novel role for the PDF-system in the regulation of egg-laying.

Structure-activity studies of Drosophila adipokinetic hormone (AKH) by a cellular expression system of dipteran AKH receptors.

Structure-activity studies for the adipokinetic hormone receptor of insects were for the first time performed in a cellular expression system. A series of single amino acid replacement analogues for the endogenous adipokinetic hormone of Drosophila melanogaster (pGlu-Leu-Thr-Phe-Ser-Pro-Asp-Trp-NH(2)) were screened for activity with a bioluminescence cellular assay, expressing the G-protein coupled receptor. For this series of peptide analogues, one amino acid of the N-terminal tetrapeptide was successively replaced by alanine, while those of the C-terminal tetrapeptide were successively substituted by glycine; other modifications included the blocked N- and C-termini that were replaced by an acetylated alanine and a hydroxyl group, respectively. The analogue series was tested on the AKH receptors of two dipteran species, D. melanogaster and Anopheles gambiae. The blocked termini of the AKH peptide probably play a minor role in receptor interaction and activation, but are considered functionally important elements to protect the peptide against exopeptidases. In contrast, the amino acids at positions 2, 3, 4 and 5 from the N-terminus all seem to be crucial for receptor activation. This can be explained by the potential presence of a ß-strand in this part of the peptide that interacts with the receptor. The inferred ß-strand is probably followed by a ß-turn in which the amino acids at positions 5-8 are involved. In this ß-turn, the residues at positions 6 and 8 seem to be essential, as their substitutions induce only a very low degree of receptor activation. Replacement of Asp(7), by contrast, does not influence receptor activation at all. This implies that its side chain is folded inside the ß-turn so that no interaction with the receptor occurs.

A pharmacological study of NLP-12 neuropeptide signaling in free-living and parasitic nematodes.

NLP-12a and b have been identified as cholecystokinin/sulfakinin-like neuropeptides in the free-living nematode Caenorhabditis elegans. They are suggested to play an important role in the regulation of digestive enzyme secretion and fat storage. This study reports on the identification and characterization of an NLP-12-like peptide precursor gene in the rat parasitic nematode Strongyloides ratti. The S. ratti NLP-12 peptides are able to activate both C. elegans CKR-2 receptor isoforms in a dose-dependent way with affinities in the same nanomolar range as the native C. elegans NLP-12 peptides. The C-terminal RPLQFamide sequence motif of the NLP-12 peptides is perfectly conserved between free-living and parasitic nematodes. Based on systemic amino acid replacements the Arg-, Leu- and Phe- residues appear to be critical for high-affinity receptor binding. Finally, a SAR analysis revealed the essential pharmacophore in C. elegans NLP-12b to be the pentapeptide RPLQFamide.

Deorphanizing g protein-coupled receptors by a calcium mobilization assay.

G protein-coupled receptors (GPCRs) comprise one of the largest families of transmembrane proteins involved in signal transduction of diverse external stimuli and represent the most successful target class in drug discovery. The availability of genome sequences in the postgenomic era has paved the way for in silico identification of novel GPCR family members based upon sequence similarity. Consequently, newly discovered receptors are by definition orphan GPCRs with no known ligand, and their functional characterization now poses a major challenge. Over the years, advances in understanding of GPCR biology have led to the development of cell-based assay systems that link orphan GPCRs to their activating ligand(s) in high-throughput format (reverse pharmacology). Many of these technologies monitor important steps in the GPCR activation cycle such as the accumulation of secondary messenger molecules (e.g., cAMP, calcium). In this chapter, we present a calcium mobilization assay in mammalian cells to detect changes in intracellular calcium concentration upon receptor activation by the use of a fluorescent probe. This is currently one of the most frequently used assay systems for GPCR deorphanization.

C. elegans homologs of insect clock proteins: a tale of many stories.

As a consequence of the Earth's axial rotation, organisms display daily recurring rhythms in behavior and biochemical properties, such as hormone titers. The neuronal system controlling such changes is best studied in the fruit fly Drosophila melanogaster. In the nematode worm Caenorhabditis elegans, most homologs of these genes function in the heterochronic pathway controlling the (timing of) developmental events. Recent data indicate that in the worm at least one of the genes involved in developmental timing is also active in circadian rhythm control, thereby opening up new perspectives on a central (neuronal) timer interfering with many processes. Also, new neuropeptidergic clock homologs have been identified in nematodes, supporting the idea of a broad range of clock-regulated targets. We will describe the current knowledge on homologous clock genes in C. elegans with a focus on the recently discovered pigment dispersing factor gene homologs. Similarities between developmental and daily timing are discussed.

Gonadotropin-releasing hormone and adipokinetic hormone signaling systems share a common evolutionary origin.

Gonadotropin-releasing hormone (GnRH) is a critical and central hormone that regulates vertebrate reproduction. The high conservation of GnRH signaling within the chordates (deuterostomians) raises the important question as to whether its appearance might date back prior to the divergence of protostomian and deuterostomian lineages, about 700 million years ago. This leads to several important questions regarding the evolution of the GnRH family. Has GnRH been retained in most protostomian lineages? And was regulation of reproduction already a function of ancestral GnRH? The first question can undoubtedly be answered affirmatively since several GnRH-like sequences have been found in wide variety of protostomian and deuterostomian phyla. However, based on their different primary functions in different phyla - which implies a less unanimous answer on the second question - consistency in the nomenclature of this peptide family has been lost. A comparative and phylogenetic approach shows that the ecdysozoan adipokinetic hormones (AKHs), lophotrochozoan GnRHs and chordate GnRHs are structurally related and suggests that they all originate from a common ancestor. This review supports the view that the AKH-GnRH signaling system probably arose very early in metazoan evolution, prior to the divergence of protostomians and deuterostomians.

Coevolution of neuropeptidergic signaling systems: from worm to man.

Despite the general knowledge and repeated predictions of peptide G protein-coupled receptors following the elucidation of the Caenorhabditis elegans genome in 1998, only a few have been deorphanized so far. This was attributed to the apparent lack of coevolution between (neuro)peptides and their cognate receptors. To resolve this issue, we have used an in silico genomic data mining tool to identify the real putative peptide GPCRs in the C. elegans genome and then made a well-considered selection of orphan peptide GPCRs. To maximize our chances of a successful deorphanization, we adopted a combined reverse pharmacology approach. At this moment, we have successfully uncovered four C. elegans neuropeptide signaling systems that support the theory of receptor-ligand coevolution. All four systems are extremely well conserved within nematodes and show a high degree of similarity with their vertebrate and arthropod counterparts. Our data indicate that these four neuropeptide signaling systems have been well conserved during the course of evolution and that they were already well established prior to the divergence of protostomes and deuterostomes.

Approaches to identify endogenous peptides in the soil nematode Caenorhabditis elegans.

The transparent soil nematode Caenorhabditis elegans can be considered an important model organism due to its ease of cultivation, suitability for high-throughput genetic screens, and extremely well-defined anatomy. C. elegans contains exactly 959 cells that are ordered in defined differentiated tissues. Although C. elegans only possesses 302 neurons, a large number of similarities among the neuropeptidergic signaling pathways can be observed with other metazoans. Neuropeptides are important messenger molecules that regulate a wide variety of physiological processes. These peptidergic signaling molecules can therefore be considered important drug targets or biomarkers. Neuropeptide signaling is in the nanomolar range, and biochemical elucidation of individual peptide sequences in the past without the genomic information was challenging. Since the rise of many genome-sequencing projects and the significant boost of mass spectrometry instrumentation, many hyphenated techniques can be used to explore the "peptidome" of individual species, organs, or even cell cultures. The peptidomic approach aims to identify endogenously present (neuro)peptides by using liquid chromatography and mass spectrometry in a high-throughput way. Here we outline the basic procedures for the maintenance of C. elegans nematodes and describe in detail the peptide extraction procedures. Two peptidomics strategies (off-line HPLC-MALDI-TOF MS and on-line 2D-nanoLC-Q-TOF MS/MS) and the necessary instrumentation are described.