Geert Baggerman

Peptidomics of the larval Drosophila melanogaster central nervous system

Neuropeptides regulate most, if not all, biological processes in the animal kingdom, but only seven have been isolated and sequenced from Drosophila melanogaster. In analogy with the proteomics technology, where all proteins expressed in a cell or tissue are analyzed, the peptidomics approach aims at the simultaneous identification of the whole peptidome of a cell or tissue,i.e. all expressed peptides with their posttranslational modifications. Using nanoscale liquid chromatography combined with tandem mass spectrometry and data base mining, we analyzed the peptidome of the larval Drosophila central nervous system at the amino acid sequence level. We were able to provide biochemical evidence for the presence of 28 neuropeptides using an extract of only 50 larval Drosophila central nervous systems. Eighteen of these peptides are encoded in previously cloned or annotated precursor genes, although not all of them were predicted correctly. Eleven of these peptides were never purified before. Eight other peptides are entirely novel and are encoded in five different, not yet annotated genes. This neuropeptide expression profiling study also opens perspectives for other eukaryotic model systems, for which genome projects are completed or in progress.

Sulfakinins reduce food intake in the desert locust, Schistocerca gregaria

In vertebrates, the peptides cholecystokinin (CCK), neuropeptide Y, galanin, and bombesin are known to be involved in the control of food intake. We report here that insect sulfakinins, peptides which display substantial sequence similarities with the vertebrate gastrin/CCK peptide family, significantly inhibit food uptake in fifth instar nymphs of the locust, Schistocerca gregaria. Upon injection of Lom-sulfakinin, a neuropeptide present in the corpus cardiacum of locusts, food intake was significantly reduced in a dose-dependent manner within a fixed 20 min time period. The induced effect ranged from 13% inhibition (10 pmol of injected peptide) to over 50% inhibition at 1 nmol. Other naturally occurring sulfakinins from different insect species also elicited this satiety effect. Analogous to the satiety effect of CCK in vertebrates, the sulfate group is required for activity. No effect on the palptip resistance was found after injection with sulfakinin. Therefore it seems unlikly that sulfakinins reduce food intake by decreasing the sensitivity of the taste receptors.

Neuropeptidomic analysis of the brain and thoracic ganglion from the Jonah crab, Cancer borealis

Mass spectrometric methods were applied to determine the peptidome of the brain and thoracic ganglion of the Jonah crab (Cancer borealis). Fractions obtained by high performance liquid chromatography were characterized using MALDI-TOF MS and ESI-Q-TOF MS/MS. In total, 28 peptides were identified within the molecular mass range 750-3000Da. Comparison of the molecular masses obtained with MALDI-TOF MS with the calculated molecular masses of known crustacean peptides revealed the presence of at least nine allatostatins, three orcokinin precursor derived peptides, namely FDAFTTGFGHS, [Ala(13)]-orcokinin, and [Val(13)]-orcokinin, and two kinins, a tachykinin-related peptide and four FMRFamide-related peptides. Eight other peptides were de novo sequenced by collision induced dissociation on the Q-TOF system and yielded AYNRSFLRFamide, PELDHVFLRFamide or EPLDHVFLRFamide, APQRNFLRFamide, LNPFLRFamide, DVRTPALRLRFamide, and LRNLRFamide, which belong to the FMRFamide related peptide family, as well as NFDEIDRSGFA and NFDEIDRSSFGFV, which display high sequence similarity to peptide sequences within the orcokinin precursor of Orconectes limosus. Our paper is the first (neuro)peptidomic analysis of the crustacean nervous system.

Characterization of an orphan G protein-coupled receptor localized in the dorsal root ganglia reveals adenine as a signaling molecule

The cloning of novel G protein-coupled receptors and the search for their natural ligands, a process called reverse pharmacology, is an excellent opportunity to discover novel hormones and neurotransmitters. Based on a degenerate primer approach we have cloned a G protein-coupled receptor whose mRNA expression profile indicates highest expression in the dorsal root ganglia, specifically in the subset of small neurons, suggesting a role in nociception. In addition, moderate expression was found in lung, hypothalamus, peripheral blood leukocytes, and ovaries. Guided by a receptor-activation bioassay, we identified adenine as the endogenous ligand, which activated the receptor potently and with high structural stringency. Therefore, we propose to name this receptor as the adenine receptor. Hormonal functions have already been demonstrated for adenine derivatives like 6-benzylaminopurine in plants and 1-methyladenine in lower animals. Here, we demonstrate that adenine functions as a signaling molecule in mammals. This finding adds a third family besides P1 and P2 receptors to the class of purinergic receptors.

Gel-Based Versus Gel-Free Proteomics: A Review

With the sequencing of the genome of over 150 organisms, the field of biology has been revolutionised. Instead of studying one gene or protein at the time, it is now possible to study the effect of physiological or pathological changes on the expression of all genes or proteins in the organism. Proteomics aims at the simultaneous analysis of all proteins expressed by a cell, tissue or organism in a specific physiological condition. Because proteins are the effector molecules in all organisms, it is evident that changes in the physiological condition of an organism will be reflected by changes in protein expression and/or processing. Since the formulation of the concept of proteomics in the mid 90s proteomics has relied heavily on 2 dimensional gel electrophoresis (2DGE) for the separation and visualization of proteins. 2DGE, however, has a number of inherent drawbacks. 2DGE is costly, fairly insensitive to low copy proteins and cannot be used for the entire proteome. Therefore, over the years, several gel-free proteomics techniques have been developed to either fill the gaps left by 2DGE or to entirely abolish the gel based techniques. This review summarizes the most important gel-free and gel-based proteomics techniques and compares their advantages and drawbacks.

Proteomics in Drosophila melanogaster: first 2D database of larval hemolymph proteins

A proteomic approach was used for the identification of larval hemolymph proteins of Drosophila melanogaster. We report the initial establishment of a two-dimensional gel electrophoresis reference map for hemolymph proteins of third instar larvae of D. melanogaster. We used immobilized pH gradients of pH 4-7 (linear) and a 12-14% linear gradient polyacrylamide gel. The protein spots were silver-stained and analyzed by nanoLC-Q-Tof MS/MS (on-line nanoscale liquid chromatography quadrupole time of flight tandem mass spectrometry) or by Matrix assisted laser desorption time of flight MS (MALDI-TOF MS). Querying the SWISSPROT database with the mass spectrometric data yielded the identity of the proteins in the spots. The presented proteome map lists those protein spots identified to date. This map will be updated continuously and will serve as a reference database for investigators, studying changes at the protein level in different physiological conditions.

Defective processing of neuropeptide precursors in Caenorhabditis elegans lacking proprotein convertase 2 (KPC‐2/EGL‐3): mutant analysis by mass spectrometry

Biologically active peptides are synthesized as larger inactive proprotein peptide precursors which are processed by the concerted action of a cascade of enzymes. Among the proprotein convertases, PC2 is widely expressed in neuro‐endocrine tissues and has been proposed to be the major convertase involved in the biosynthesis of neuropeptides. In this study, we have examined the role of the Caenorhabditis elegans orthologue PC2/EGL‐3 in the processing of proprotein peptide precursors. We recently isolated and identified 60 endogenous peptides in the nematode C. elegans by two‐dimensional nanoscale liquid chromatography – quadrupole time‐of‐flight tandem mass spectrometry. In the present study, we compare the peptide profile of different C. elegans strains, including PC2/EGL‐3 mutants. For this purpose, we used an offline approach in which HPLC fractions are analysed by a matrix‐assisted laser desorption ionisation – time of flight mass spectrometer. This differential peptidomic approach unambiguously provides evidence for the role of PC2/EGL‐3 in the processing of FMRFamide‐like peptide (FLP) precursors and neuropeptide‐like protein (NLP) precursors in nematodes.

Identification in Drosophila melanogaster of the invertebrate G protein-coupled FMRFamide receptor

We here describe the cloning and characterization of the functionally active Drosophila melanogaster (Drm) FMRFamide receptor, which we designated as DrmFMRFa-R. The full-length ORF of a D. melanogaster orphan receptor, CG 2114 (Berkeley Drosophila Genome Project), was cloned from genomic DNA. This receptor is distantly related to mammalian thyroid-stimulating hormone-releasing hormone receptors and to a set of Caenorhabditis elegans orphan receptors. An extract of 5,000 central nervous systems from the related but bigger flesh fly, Neobellieria bullata (Neb), was used to screen cells expressing the orphan receptor. Successive purification steps, followed by MS, revealed the sequence of two previously uncharacterized endogenous peptides, APPQPSDNFIRFamide (Neb-FIRFamide) and pQPSQDFMRFamide (Neb-FMRFamide). These are reminiscent of other insect FMRFamide peptides, having neurohormonal as well as neurotransmitter functions. Nanomolar concentrations of the Drm FMRFamides (DPKQDFMRFamide, TPAEDFMRFamide, SDNFMRFamide, SPKQDFMRFamide, and PDNFMRFamide) activated the cognate receptor in a dose-dependent manner. To our knowledge, the cloned DrmFMRFa-R is the first functionally active FMRFamide G protein-coupled receptor described in invertebrates to date.

In Silico Identification of New Secretory Peptide Genes in Drosophila melanogaster

Bioactive peptides play critical roles in regulating most biological processes in animals. The elucidation of the amino acid sequence of these regulatory peptides is crucial for our understanding of animal physiology. Most of the (neuro)peptides currently known were identified by purification and subsequent amino acid sequencing. With the entire genome sequence of some animals now available, it has become possible to predict novel putative peptides. In this way, BLAST (Basic Local Alignment Searching Tool) analysis of the Drosophila melanogaster genome has allowed annotation of 36 secretory peptide genes so far. Peptide precursor genes are, however, poorly predicted by this algorithm, thus prompting an alternative approach described here. With the described searching program we scanned the Drosophila genome for predicted proteins with the structural hallmarks of neuropeptide precursors. As a result, 76 additional putative secretory peptide genes were predicted in addition to the 43 annotated ones. These putative (neuro)peptide genes contain conserved motifs reminiscent of known neuropeptides from other animal species. Peptides that display sequence similarities to the mammalian vasopressin, atrial natriuretic peptide, and prolactin precursors and the invertebrate peptides orcokinin, prothoracicotropic hormones, trypsin modulating oostatic factor, and Drosophila immune induced peptides (DIMs) among others were discovered. Our data hence provide further evidence that many neuropeptide genes were already present in the ancestor of Protostomia and Deuterostomia prior to their divergence. This bioinformatic study opens perspectives for the genome-wide analysis of peptide genes in other eukaryotic model organisms.

Peptidomics: The integrated approach of MS, hyphenated techniques and bioinformatics for neuropeptide analysis

MS is currently one of the most important analytical techniques in biological and medical research. ESI and MALDI launched the field of MS into biology. The performance of mass spectrometers increased tremendously over the past decades. Other technological advances increased the analytical power of biological MS even more. First, the advent of the genome projects allowed an automated analysis of mass spectrometric data. Second, improved separation techniques, like nanoscale HPLC, are essential for MS analysis of biomolecules. The recent progress in bioinformatics is the third factor that accelerated the biochemical analysis of macromolecules. The first part of this review will introduce the basics of these techniques. The field that integrates all these techniques to identify endogenous peptides is called peptidomics and will be discussed in the last section. This integrated approach aims at identifying all the present peptides in a cell, organ or organism (the peptidome). Today, peptidomics is used by several fields of research. Special emphasis will be given to the identification of neuropeptides, a class of short proteins that fulfil several important intercellular signalling functions in every animal. MS imaging techniques and biomarker discovery will also be discussed briefly.