ena-STUDY-IRSTEA-13-12-2013-14:09:56:598-44
Proteogenomics of Gammarus fossarum
Organisms were sampled from a field population located in Eastern Central France. Because our focus on proteins involved in reproduction, only sexually mature organisms were chosen by selecting the ones in pre-copulatory mate guarding. We first established a database of nucleic acid-derived protein coding sequences. For this, we extracted total RNAs from four key tissues: the male and female reproductive tissues, the cephalon where several neuroendocrine glands are located and the hepatopancreatic caeca, involved in xenobiotics detoxification and energy acquisition (digestive enzyme secretion, food absorption and nutriment storage). Equal amounts of RNAs from each tissue were pooled together and sequenced in a single run of the Illumina next-generation sequencing approach. The resulting nucleic acid information was used to create a protein database that could be used for proteogenomic identification of proteins. For this, each mRNA contig was systematically translated into the six possible reading frames, thus for considerating all possible putative ORFs. Tandem mass spectrometry data could be assigned with this database. The protein catalogue established from experimental pieces of evidence allows selecting the appropriate reading direction and frame of each protein-coding mRNA contig. We recorded a large dataset on the proteomes from three tissues (reproductive tissues and the cephalon) by resolving each sample by OFFGEL electrophoresis (24 fractions per sample). Each experiment was conducted in triplicates for reproductive tissues and duplicates for the cephalon, resulting in 192 peptide complex mixtures. These samples were analyzed by nanoLC-MS/MS with a high resolution LTQ-Orbitrap XL hybrid mass spectrometer (ThermoFisher). We also focused our attention on the proteome dynamics of the male reproductive tissue analyzed at seven different spermatogenesis stages (pre-copulatory mate guarding, Day 0, Day 1, Day 2, Day 3, Day 4 and Day7) on five biological replicates. Protein extracts from the testis samples were subjected to a short SDS-PAGE electrophoresis in order to get a single polyacrylamide band per proteome. The 35 samples were in-gel trypsic digested and the resulting peptide mixtures were analyzed by nanoLC-MS/MS.
Organisms were sampled from a field population located in Eastern Central France. Because our focus on proteins involved in reproduction, only sexually mature organisms were chosen by selecting the ones in pre-copulatory mate guarding. We first established a database of nucleic acid-derived protein coding sequences. For this, we extracted total RNAs from four key tissues: the male and female reproductive tissues, the cephalon where several neuroendocrine glands are located and the hepatopancreatic caeca, involved in xenobiotics detoxification and energy acquisition (digestive enzyme secretion, food absorption and nutriment storage). Equal amounts of RNAs from each tissue were pooled together and sequenced in a single run of the Illumina next-generation sequencing approach. The resulting nucleic acid information was used to create a protein database that could be used for proteogenomic identification of proteins. For this, each mRNA contig was systematically translated into the six possible reading frames, thus for considerating all possible putative ORFs. Tandem mass spectrometry data could be assigned with this database. The protein catalogue established from experimental pieces of evidence allows selecting the appropriate reading direction and frame of each protein-coding mRNA contig. We recorded a large dataset on the proteomes from three tissues (reproductive tissues and the cephalon) by resolving each sample by OFFGEL electrophoresis (24 fractions per sample). Each experiment was conducted in triplicates for reproductive tissues and duplicates for the cephalon, resulting in 192 peptide complex mixtures. These samples were analyzed by nanoLC-MS/MS with a high resolution LTQ-Orbitrap XL hybrid mass spectrometer (ThermoFisher). We also focused our attention on the proteome dynamics of the male reproductive tissue analyzed at seven different spermatogenesis stages (pre-copulatory mate guarding, Day 0, Day 1, Day 2, Day 3, Day 4 and Day7) on five biological replicates. Protein extracts from the testis samples were subjected to a short SDS-PAGE electrophoresis in order to get a single polyacrylamide band per proteome. The 35 samples were in-gel trypsic digested and the resulting peptide mixtures were analyzed by nanoLC-MS/MS.