Gel-free mass spectrometry analysis of Drosophila melanogaster heads
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Velos
ORGANISM(S): Drosophila Melanogaster (fruit Fly)
TISSUE(S): Head
SUBMITTER: 
Jana Aradska
LAB HEAD: Gert Lubec
PROVIDER: PXD001712 | Pride | 2015-07-27
REPOSITORIES: Pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| Lubeca03.mgf | Mgf | |||
| Lubeca03.pride.mgf.gz | Mgf | |||
| Lubeca04.mgf | Mgf | |||
| Lubeca04.pride.mgf.gz | Mgf | |||
| Lubeca05.mgf | Mgf |
Items per page: 5 1 - 5 of 351 |
Publications
Gel-free mass spectrometry analysis of Drosophila melanogaster heads.
Aradska Jana J Bulat Tanja T Sialana Fernando J FJ Birner-Gruenberger Ruth R Erich Buchner B Lubec Gert G Lubec Gert G
Proteomics 20150910 19
Membrane proteins play key roles in several fundamental biological processes such as cell signalling, energy metabolism and transport. Despite the significance, these still remain an under-represented group in proteomics datasets. Herein, a bottom-up approach to analyse an enriched membrane fraction from Drosophila melanogaster heads using multidimensional liquid chromatography (LC) coupled with tandem-mass spectrometry (MS/MS) that relies on complete solubilisation and digestion of proteins, is ...[more]
Similar Datasets
Project description:Previous work has shown that a series of honey bee brain proteins were probably linked to the proboscis extension reflex (PER) , a model studying insect behaviour. Herein, it was the aim of the study to generate a “synaptic proteome” and to investigate which proteins were paralleling olfactory conditioning. To address this information, we applied a non-sophisticated olfactory conditioning model using the proboscis extension reflex (PER) in the honeybee (Apis mellifera). Foraging honeybees were used in the study and three groups were formed, animals on water control, sucrose (PER) and olfactory conditioned (PER-conditioned) using 2-octanone. A synaptosomal preparation of honey bee brain for quantitative proteomics using stable isotope labelling (TMT 10plex) was performed. In addition a synaptosomal brain proteome was generated. Protein levels that were modified during olfactory conditioning were associated with “SNARE interactions in vesicular transport” (BET1 and VAMP7), ABC transporters, and fatty acid degradation pathways.. A honey bee synaptosomal proteome dataset including neurotransmitter receptors and transporters was generated.
2019-05-27 | PXD011217 | Pride
Project description:Expression level polymorphisms (ELPs) often result in cis-acting expression quantitative trait loci (cis-eQTL), which are important QTL and association mapping tools and account significantly for phenotypic variability. Generally, it is assumed that such stably heritable ELP represent regulatory element polymorphisms in the respective genes. However, comprehensive genome-wide analyses linking expression level, regulatory sequence and gene structure variation are missing, preventing definite verification of this assumption. Here we analyzed heritability of ELP observed between Arabidopsis thaliana accessions Eil-0 and Lc-0 by comparing genotyped recombinant inbred lines (RIL) to their parents in microarray analyses. Keywords: expression level polymorphism, Arabidopsis thaliana accessions, recombinant inbred lines In order to analyze expression level polymorphisms between the accessions Eil-0 and Lc-0, three independently grown seedling pools were analyzed by two color co-hybridization of the labeled cDNAs in dyeswap experiments, giving a total of six slides. For the analysis of gene expression in seedlings of 7 different RILs, each RIL sample was co-hybridized with each parent (Eil-0 and Lc-0) in a dyeswap, resulting in two slides per parent vs. RIL comparison. The total number of slides in this study was 34.
2010-05-16 | E-GEOD-13628 | biostudies-arrayexpress
Project description:The perinuclear theca (PT) is a highly condensed, largely insoluble protein structure that surrounds the nucleus of eutherian spermatozoa. While this structure is known to be important for fertilization, little is known regarding its proteomic composition. Recent reports have indicated that the PT unexpectedly houses several somatic proteins, such as core histones, which may be important post-fertilization during re-modelling of the male pronucleus. To explore the proteomic composition of the PT we performed the first in depth, label-free proteomic characterization of the PT of boar spermatozoa. To do this, a unique subcellular fractionation protocol was first performed to isolate the PT and increase our ability to detect lowly abundant sperm proteins. Through the use of this subcellular fractionation technique we were able to quantify 1802 proteins, a result that represents unparalleled depth of coverage for the boar sperm proteome and exceeds the entire known proteome of the Sus scrofa species so far. In the PT structure itself we identified 813 proteins and confirmed the presence of previously characterized PT proteins including the core histones H2A, H2B, H3 and H4, as well as Ras-related protein Rab-2A (RAB2A) and Rab-2B (RAB2B) amongst other RAB proteins. In addition to these previously characterized PT proteins, our data also revealed that the PT is replete in proteins critical for sperm-egg fusion including: Izumo sperm-egg fusion proteins 1-4 (IZUMO1-4) and phosphoinositide phospholipase C (PLCZ1).
2022-03-09 | PXD030020 | Pride
Project description:Chinese hamster ovary (CHO) cells have been widely employed for production of recombinant proteins (RP) for research and therapeutic purposes. Indeed, this expression system was used for production of most of approved antibodies (84%) in 2015-2018 period. The success of this cell line for RP production relies on several advantages that comprise but are not limited to a safety viral profile, human compatible glycosylation, development of specific culture mediums and supplements, availability of sub-lines with different capabilities and the improvement in design of DNA vectors and selection strategies that allows to obtain higher producer clones in an easier way. Given the high importance of this cell line, a deeper knowledge of their biology through genomic, transcriptomic, proteomic and metabolomic studies has been gained in the last years. These studies have aid in the exploration of molecular and cellular mechanisms that could explain cell behavior and to propose new targets to improve production and quality of RP. Some of these transcriptomic and proteomic profiles have been acquired under low temperature, butyrate addition, hyperosomotic pressure, protein degradation phenotypes, cellular engineering, production stability and productive phenotypes. In case of productive phenotypes, several whole cell proteomic studies have been previously reported, where cell populations secreting fusion proteins, monoclonal antibodies (mAb) and antibody fragments, at different specific productivity (Qp), have been compared. However, because of this information has been obtained from whole cell homogenates, highlighted categories represent abundant proteins and have limited coverage of proteome from some organelles such as the classical secretory pathway. Due to the central role of this pathway in protein and lipid metabolism, organelle biogenesis, cell cycle, apoptosis and proliferation, and to be recognized as a bottleneck for protein secretion in mammalian cells, its characterization through subcellular proteomics represents a promising strategy for identification of key targets. In fact, over-expression of some proteins from endoplasmic reticulum and Golgi Apparatus has increased the Qp of HEK293 and CHO cells producing different RP. Recently, proteomic reports of subcellular compartments from a variety of cells have been extensively used to study the protein composition of organelles, protein dynamics and discovering of novel proteins involved in the secretion pathway. Thus, this approach was applied to the secretory pathway of CHO cells to identify novel proteins linked to Qp in the present study. Differential proteomics from all remaining compartments was also carried out, giving new lights over other cellular mechanisms associated to the higher producer phenotype. The novel differentially expressed proteins (DEP) found will improve the engineering strategies of CHO cells that will have a positive impact on titer and quality of RP. Cell lines CHO DP-12 clone #1933 CRL-12444 and clone #1934 CRL-12445, which secrete a mAb against human interleukin 8 (IL-8), were used as cell models of recombinant CHO cell lines producing the same RP at different Qp. Our goal is that the processing of raw proteomics data and the identification and classification of DEP belonging to the classical secretory pathway will allow to propose a model by which the increase in the productivity of CHO cells has been associated with the deregulation of proteins from this pathway that show differential expression among the clones under study. This strategy of subcellular proteomics allowed us to identify a large number of new targets and cell pathways with respect to previous whole cell proteomic studies, which can improve the understanding of the molecular mechanisms behind RP production and provide the basis to design new sub-lines with stronger capabilities.
2021-05-04 | PXD021014 | Pride
Project description:Chinese hamster ovary (CHO) cells have been widely employed for production of recombinant proteins (RP) for research and therapeutic purposes. Indeed, this expression system was used for production of most of approved antibodies (84%) in 2015-2018 period. The success of this cell line for RP production relies on several advantages that comprise but are not limited to a safety viral profile, human compatible glycosylation, development of specific culture mediums and supplements, availability of sub-lines with different capabilities and the improvement in design of DNA vectors and selection strategies that allows to obtain higher producer clones in an easier way. Given the high importance of this cell line, a deeper knowledge of their biology through genomic, transcriptomic, proteomic and metabolomic studies has been gained in last years. These studies have aid in the exploration of molecular and cellular mechanisms that could explain cell behavior and to propose new targets for improved RP production and quality. A special emphasis has been placed on proteomic characterization of subcellular compartments over cellular homogenates, due to some advantages that fractionation techniques offer over the classical approach, which include obtaining datasets that are easier to process and understand. Subcellular proteomics allows monitoring of proteins that shuttle between different compartments, quantification of lower abundance proteins, increase in number of identified proteins, elucidation of function and regulation of proteins, and unraveling of organelle dynamics. The use of subcellular fractionation for proteomic characterization of organelles from CHO cells has been limited to the isolation of one or few organelles in an adherent phenotype, and only few fractionation protocols have been reported for these cells in their suspension format. The high cross-contamination of some isolated fractions due to insufficient fractionation steps, prevents adopting some of these protocols for applications such as proteomics. Thus, since this approach has not been addressed before for the proteomic characterization of CHO cell organelles, subcellular proteomics was harnessed in the present study to provide identification and quantification of proteins from subcellular compartments obtained by differential and isopycnic centrifugation of this cell line. Although several cellular processes and organelles play an important role during expression and secretion of RP, the classical secretion pathway has been recognized as a bottleneck for post-translational modifications, transport, modification and secretion of proteins in mammalian cells. In fact, over-expression of some proteins from endoplasmic reticulum and Golgi Apparatus has increased the specific productivity of HEK293 and CHO cells producing different RP. Thus, in line with the paramount importance of organelles from classical secretion pathway for RP production, a novel discontinuous sucrose gradient for improved separation and enrichment of microsomes has been designed in our laboratory, and incorporated to the proteomic characterization of CHO cell organelles. CRL-12444, a DP-12 derived cell line producing a humanized monoclonal antibody against human IL-8, was used as a model of a recombinant CHO cell line during this study. Our goal is that the processing of raw proteomics data, the classification and mapping of identified proteins will allow to construct proteomic maps for most subcellular compartments, describing the molecular functions, biological processes and pathways from enriched fractions. The identification of proteins in each enriched compartment will improve the knowledge about their protein components, function and interaction, especially from those poorly represented like the proteins in the microsomes, allowing a subsequently expansion of the engineering strategies of CHO cells to increase the titer and quality of RPs.
2020-08-27 | PXD019778 | Pride
Project description:Recessive retinitis pigmentosa (RP) is often caused by nonsense mutations that lead to low mRNA levels as a result of nonsense-mediated decay. Some RP genes are expressed at detectable levels in leukocytes as well as in the retina. We designed a microarray-based method to find recessive RP genes based on low lymphoblast mRNA expression levels Experiment Overall Design: We established lymphoblast cell lines from 13 unrelated index patients with recessive RP as well all of their affected siblings (1 sibship with 4 affected members, 5 with 2 affected members, and 7 isolates) and 4 controls. RNA was isolated and hybridized on Affymetrix genechip Human Genome U133Plus2.0. After normalization, expression levels of the individual families were compared to the other samples; significance was tested using the Student t-test. The most significant suitable candidate genes were sequenced to screen for disease causing mutations and/or analyzed for segregation in the family.
2008-10-21 | E-GEOD-12086 | biostudies-arrayexpress
Project description:Human neural organoid models have become an important tool for studying neurobiology. In this work, we compared Matrigel to an N-cadherin peptide-functionalized gelatin methacryloyl hydrogel (termed GelMA-Cad) for culturing cortical neural organoids. Specifically, we compare five materials: (1) Matrigel, (2) GelMA-Cad with high crosslinker (HC), (3) GelMA-Cad with low crosslinker (LC), (4) GelMA HC and (5) GelMA LC. We profiled these organoids at the earliest stages to understand potential differences in radial glia formation.
2024-07-01 | E-MTAB-14203 | biostudies-arrayexpress
Project description:In this study the impact of protein fractionation techniques prior to LC/MS analysis was investigated on activated sludge samples derived at winter and summer condition from a full-scale wastewater treatment plant (WWTP). For reduction of the sample complexity, different fractionation techniques including RP-LC (1D-approach), SDS-PAGE and RP-LC (2D-approach) as well as RP-LC, SDS-PAGE and liquid IEF (3D-approach) were carried out before subsequent ITMS analysis. The derived spectra were identified by MASCOT search using a combination of the public UniProtKB/Swiss-Prot protein database and metagenome data from a WWTP (data are available via ProteomeXchange with identifier PXD001547). The results showed a significant increase of identified spectra, enabled by applying IEF and SDS-PAGE to the proteomic workflow. Based on meta-proteins, a core metaproteome and the corresponding taxonomic profile of the wastewater activated sludge was revealed and functional aspects using the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway library were analyzed. Hereby, the KEGG Orthology identifiers (KO numbers) of protein hits were plotted into pathway maps of the central carbon (map01200) and nitrogen metabolism (map00910). Using the 3D-approach, most proteins involved in glycolysis and citrate cycle and nearly all proteins of the nitrogen removal were identified, qualifying this approach as the most promising for future studies.
2015-07-28 | PXD001547 | Pride
Project description:Shotgun proteomics including TiO2 enrichment of phosphorylated peptides followed by reversed-phase liquid chromatography tandem mass spectrometry (RP-LC MS/MS) on lysates from glucose-stimulated INS-1E cells was used to identify glucose regulated phosphorylated proteins and signal transduction pathways.
2019-02-25 | PXD011329 | Pride
Project description:Stress survival tactics in bacteria utilize the up- and down-regulation of stress response genes. In bacteria that lack classical stress response genes for oxidative stress, other cellular systems can be used for cell survival. We used custom microarrays to study the regulation of genes in Bifidobacterium animalis ssp. lactis strains to oxidative stress to elucidate novel stress response mechanisms. Bifidobacterium cells were grown to late log phase then harvested and exposed to a sub-lethal level of hydrogen peroxide. Samples were taken at 5 and 20 mins for RNA extraction and hybridization on Affymetrix microarrays. Controls were prepared for each time point which recieved no hydrogen peroxide treatment.
2013-02-20 | E-GEOD-44382 | biostudies-arrayexpress