Project description:Quantitative comparation of the tagmata of body and head using iTRAQ based on HCD Fragmentation. The 8-plex iTRAQ Multiplex Buffer Kit (AB SCIEX, Foster City, CA) was used to label different peptide fractions, in which iTRAQ reagents dissolved in isopropyl alcohol. Another internal standard of D.melanogaster body and head mixed with 1:1 (B+H) was also used when doing iTRAQ labeling. Therefore the same amount of B (body), H1 (head), H2 (head), B+H (internal standard) peptide fractions prepared as described above were labeled by equal but different iTRAQ reagents and incubated for 5 h at room temperature. The four different labeled peptide fractions (B:113, H1:114, H2:115, B+H:116 ) were then mixed with 1:1 and dried in a speedvac followed by desalting purification using stage tip. All prepared peptides were further analyzed on an LTQ-Orbitrap Velos hybrid mass spectrometer (Thermo Electron, San Jose, CA) coupled with UPLC (nano Acquity Ultra Performance LC, Waters). For HCD raw files, the profile data was firstly centralized by ReAdW.exe in TPP and then deisotoped and deconvoluted using in-house made scripts to improve the identification rate of spectra. All MGF files were searched using Mascot 2.3 against a Drosophila melanogaster database with 24,043 entries (http://flybase.org/, release 5.4, 24,043 entries). The target-decoy based strategy was used to control the peptide false discovery rate (FDR).
Project description:Identification and annotation of all the genes in the sequenced Drosophila genome is a work in progress. Wild-type testis function requires many genes and is thus of potentially high value for the identification of transcription units. We therefore undertook a survey of the repertoire of genes expressed in the Drosophila testis by computational and microarray analysis. We generated 3141 high-quality testis expressed sequence tags (ESTs). Testis ESTs computationally collapsed into 1560 cDNA set used for further analysis. Of those, 11% correspond to named genes, and 33% provide biological evidence for a predicted gene. A surprising 47% fail to align with existing ESTs and 16% with predicted genes in the current genome release. EST frequency and microarray expression profiles indicate that the testis mRNA population is highly complex and shows an extended range of transcript abundance. Furthermore, >80% of the genes expressed in the testis showed onefold overexpression relative to ovaries, or gonadectomized flies. Additionally, >3% showed more than threefold overexpression at p <0.05. Surprisingly, 22% of the genes most highly overexpressed in testis match Drosophila genomic sequence, but not predicted genes. These data strongly support the idea that sequencing additional cDNA libraries from defined tissues, such as testis, will be important tools for refined annotation of the Drosophila genome. Additionally, these data suggest that the number of genes in Drosophila will significantly exceed the conservative estimate of 13,601. Keywords: other
Project description:In order to compare protein profiles across different tissues, we utilized a proteomic approach that involved the DIA acquisition mode. After quantifying the data using DIA-NN software, we successfully identified and quantified 6538 proteins from the head, gut, whole body, and muscle, respectively.