Project description:In this study we use a combination of proteomics Label-Free quantification methods to monitor protein expression changes over a time course of more than 20 hours of embryo development in Drosophila melanogaster.
Project description:Nucleosomal chromatin persists in the mature sperm of Drosophila melanogaster. Paternal epigenetic marks of repression and active transcription are found within many genes essential for embryogenesis. These marks are delivered at fertilization and are subsequently maintained in the early embryo.
Project description:A spectral library was built for Drosophila melanogaster. The spectral library allows reproducible quantification for thousands of peptides per SWATH-MS analysis.
Proteins from Drosophila melanogaster embryo, adult flies were digested with trypsin using in-gel digestion and the peptides were fractionated by high-pH reverse phase chromatography. HRM peptides were spiked into the peptides mixture and each fraction was injected on a Sciex TripleTOF 6600 mass spectrometer fitted with microflow set-up.
The resulting .wiff files were analysed using MaxQuant and Spectronaut.
Project description:Drosophila melanogaster is a well-studied genetic model organism with several large-scale transcriptome resources. Here we investigate 7,952 proteins during the fly life cycle from embryo to adult and also provide a high-resolution temporal time course proteome of 5,458 proteins during embryogenesis. We use our large scale data set to compare transcript/protein expression, uncovering examples of extreme differences between mRNA and protein abundance. In the embryogenesis proteome, the time delay in protein synthesis after transcript expression was determined. For some proteins, including the transcription factor lola, we monitor isoform specific expression levels during early fly development. Furthermore, we obtained firm evidence of 268 small proteins, which are hard to predict by bioinformatics. We observe peptides originating from non-coding regions of the genome and identified Cyp9f3psi as a protein-coding gene. As a powerful resource to the community, we additionally created an interactive web interface (http://www.butterlab.org) advancing the access to our data.
Project description:Drosophila melanogaster is a well-studied genetic model organism with several large-scale transcriptome resources. Here we investigate 7,952 proteins during the fly life cycle from embryo to adult and also provide a high-resolution temporal time course proteome of 5,458 proteins during embryogenesis. We use our large scale data set to compare transcript/protein expression, uncovering examples of extreme differences between mRNA and protein abundance. In the embryogenesis proteome, the time delay in protein synthesis after transcript expression was determined. For some proteins, including the transcription factor lola, we monitor isoform specific expression levels during early fly development. Furthermore, we obtained firm evidence of 268 small proteins, which are hard to predict by bioinformatics. We observe peptides originating from non-coding regions of the genome and identified Cyp9f3psi as a protein-coding gene. As a powerful resource to the community, we additionally created an interactive web interface (http://www.butterlab.org) advancing the access to our data.
Project description:Cytosine methylation in the genome of Drosophila melanogaster has been elusive and controversial: methylcytosine has been detected at very low levels in early embryos, but the genomic location and function of methylation has not been established. We have mapped cytosine methylation genomewide in Stage 5 Drosophila embryo DNA by combining immuno-enrichment for 5-methylcytosine, bisulfite conversion, and deep sequencing. Unlike methylation patterns observed in other eukaryotic species, methylation in Drosophila is punctate and highly strand-asymmetrical; we confirmed this by direct PCR amplification and sequencing of bisulfite-converted DNA. Despite the locally asymmetric nature of methylation, large-scale patterns of methylation are symmetric. Methylated regions make up ~1% of the genome, and within these regions methylation of individual cytosines averages 2-10%. Methylation is concentrated in specific 5-base sequence motifs that are CA- and CT-rich but depleted of guanine. It is depleted from promoters, coding sequences, and most retrotransposons, and enriched in introns and in certain simple sequence repeats containing the commonly methylated motifs. Comparison with available gene expression data indicates that methylation in a gene is associated with lower expression; the X chromosome, which is subject to gene dosage compensation, is more densely methylated than the autosomes. This study firmly establishes the presence of cytosine methylation in Drosophila; the temporal overlap of methylation with the maternal-zygotic transition raises the possibility that methylation participates in the transition to zygotic gene expression. To enrich for rare cytosine methylation in Drosophila at embryonic Stage 5 (2-3 hours post-fertilization), we enriched sonicated Stage 5 genomic DNA for methylcytosine by immunoprecipitation with antibody to 5-methylcytosine. The immunoprecipitated DNA was then bisulfite converted and Illumina sequenced to obtain direct evidence for the presence of methylation. The presence and extent of DNA methylation was confirmed by Illumina sequencing of bisulfite-converted PCR amplicons.