Peptidomics of the Larval Drosophila melanogasterCentral Nervous System

Abstract

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.The most common approach in proteomic studies is to separate and visualize as many proteins as possible of an organism, tissue, or cell, by two-dimensional electrophoresis and to subsequently identify differentially expressed proteins by mass spectrometric techniques. One of the major constraints of this technology is that proteins of a molecular mass lower than 10 kDa are generally not retained and overlooked in most of the proteomic studies. Nevertheless this mass region contains a group of very important proteins, the peptide hormones and neurotransmitters.To date, many neuropeptides have been purified from vertebrate and invertebrate sources. Peptide physiology in the pregenomic era (before the realization of the genome projects) was time-consuming as a peptide present in tissue extracts had to be purified to homogeneity prior to sequencing, synthesis, and functional analysis.Drosophila is an outstanding model system for the molecular genetics and developmental biology in higher eukaryotes. Yet, its molecular endocrinology is poorly documented because only seven neuropeptides have been identified by traditional purification. Recently, a milestone was reached with the completion of the Drosophila genome project (1). Through the BLAST program, one can screen the genome of an organism for candidate-neuropeptides based on sequence homology with known neuropeptides from other organisms. In this way, 31 (neuro)peptide genes were found in Drosophila melanogaster (2-5). These findings, however, give no information on the temporal and spatial expression nor on the physiological relevance of these (neuro)peptide genes. Often, the precursors of biologically active peptides encode several peptides. In general, these peptides can be predicted from the precursor gene as it is assume...

Peptidomic analysis of the larval Drosophila melanogaster central nervous system by two‐dimensional capillary liquid chromatography quadrupole time‐of‐flight mass spectrometry

“…Drm-SK-2 (GGDDQFDDYGHMRFamide) was the only sulfakinin that could be visualized in the 1D LC/MS experiment. 5 This peptide could not be identified in our present study. Instead, we observed and identified both the sulfated and non-sulfated form of Drm-SK-1 (FDDYGHMRFamide) (Fig.…”

“…Eighteen peptides were identified in our previous one-dimensional LC/MS analysis. 5 The remaining 20 were identified for the first time by the 2D LC/MS analysis. Figure 2 shows the distribution of the identified peptides over the different SCX salt fractions.…”

“…However, a peptide, corresponding to the first nine amino acids of DrmsNPF2 (WFGDVNKPI), has been detected and sequenced. 5 This seems to indicate that the arginine at position 10 acts as a monobasic cleavage site. This finding was confirmed in the present 2D LC/MS analysis since we identified a peptide with the sequence SPSLRLRFamide corresponding to AA 11-18 of Drm-sNPF-2 but also to AA 4-11 of Drm-sNPF-1, indicating that the arginine residue at position 3 in sNPF-1 might also be an overlooked cleaving site.…”

“…A Drosophila gene encoding five putative peptides with sequence similarities to Locusta MIP 37 has been cloned recently. 38 Prior to our first 1D LC/MS peptidomic analysis, 5 no Drosophila MIP was ever isolated or sequenced from Drosophila tissue. We were able to detect and sequence two of the five predicted peptides in the larval brain extract.…”

“…4 In a previous study we used a capillary liquid chromatography/tandem mass spectrometric approach to identify neuropeptides in the central nervous systems (CNSs) of the fruit fly Drosophila melanogaster. 5 We were able to identify 28 peptides originating from 17 different precursors. Only seven of these had been isolated and sequenced before.…”