Abstract
Because of their ecological importance, amphipod crustacea are employed worldwide as test species in environmental risk assessment. Although proteomics allows new insights into the molecular mechanisms related to the stress response, such investigations are rare for these organisms because of the lack of comprehensive protein sequence databases. Here, we propose a proteogenomic approach for identifying specific proteins of the freshwater amphipod Gammarus fossarum, a keystone species in European freshwater ecosystems. After deep RNA sequencing, we created a comprehensive ORF database. We identified and annotated the most relevant proteins detected through a shotgun tandem mass spectrometry analysis carried out on the proteomes from three major tissues involved in the organism's reproductive function: the male and female reproductive systems, and the cephalon, where different neuroendocrine glands are present. The 1,873 mass-spectrometry-certified proteins represent the largest crustacean proteomic resource to date, with 218 proteins being lineage specific. Comparative proteomics between the male and female reproductive systems indicated key proteins with strong sexual dimorphism. Protein expression profiles during spermatogenesis at seven different stages highlighted the major gammarid proteins involved in the different facets of reproduction.
Highlights
Next-generation proteomics, relying on ultra-rapid and subparts-per-million mass spectrometry analyzers, is able to offer in-depth insights into the molecular players sustaining the physiology of complex organisms
General Proteogenomic Strategy for Discovering Key Proteins Involved in Reproduction from a Nonmodel Complex Organism—To represent as accurately as possible the real ecology of gammarids, we sampled heterogeneously sized organisms from a field population located in eastern central France
As in previous proteogenomic studies, tandem mass spectrometry data could be assigned with such a database [23]
Summary
Sampling and Maintenance of Animals—The amphipods were sampled at La Tour du Pin (latitude, 45°569Ј442Љ; longitude, 5°459Ј115Љ), upstream of the Bourbre River (mid-eastern France). For RNA library preparation, each tissue was collected as one tissue-type sample using 11, 9, 57, and 173 organisms for cephalons, caeca, oocytes, and testes, respectively. Peptide samples (2 l) were loaded and desalted online on a reverse-phase precolumn (C18 PepMap 100 column, LC Packings) and resolved on a nanoscale C18 PepMapTM 100 capillary column (LC Packings) at a flow rate of 0.3 l/min with a gradient of CH3CN, 0.1% formic acid prior to injection into the ion trap mass spectrometer. Statistical Data Treatment for Comparative Proteomics—Spectral counts (the number of spectra recorded per protein) were extracted from the spectra-to-peptide dataset, and the normalized spectral abundance factor for each protein was calculated as described previously [31]. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org/) via the PRIDE partner repository [32] with the dataset identifier PXD000576
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
