Seeds of Pisum sativum L. cv Alaska Early: non-aged controls vs. Aged
Ontology highlight
ABSTRACT:
ORGANISM(S): Pisum sativum
SUBMITTER: Oscar Lorenzo
PROVIDER: E-GEOD-24864 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Recent studies in non-human model systems have shown therapeutic potential of modified mRNA (modRNA) treatments for lysosomal storage diseases. Here, we assessed the efficacy of a modRNA treatment to restore the expression of the α-galactosidase (GLA) gene in a human cardiac model generated from induced-pluripotent stem cell-derived from two patients with Fabry disease. In line with the clinical phenotype, cardiomyocytes from Fabry patient’s induced pluripotent stem cells show accumulation of the glycosphinolipid Globotriaosylceramide (GB3), which is an α-galactosidase substrate. Further, the patient-specific cardiomyocytes have significant upregulation of lysosomal associated proteins. Upon modRNA treatment, a subset of lysosomal proteins were partially restored to wildtype levels, implying the rescue of the molecular phenotype associated with the Fabry genotype. Importantly, a significant reduction of GB3 levels was observed in GLA modRNA treated cardiomyocytes demonstrating that α-galactosidase enzymatic activity was restored. Together, our results validate the utility of patient IPSC-derived cardiomyocytes as a model to study disease processes in Fabry disease and the therapeutic potential of GLA modRNA treatment to reduce GB3 accumulation in the heart.
2023-07-20 | PXD038361 | Pride
Project description:Hybrid quadrupole time-of-flight (QTOF) is one of the two major mass spectrometric technologies used in proteomics. Although based on simple fundamental principles, it has over the last decades greatly evolved in terms of achievable resolution, mass accuracy and dynamic range. The Bruker impact platform of QTOF instruments takes advantage of these developments and here we develop and evaluate the impact II for shotgun proteomics applications. Adaption of our heated LC system achieved very narrow peptide elution peaks. The impact II is equipped with a new collision cell with both axial and radial ion ejection, more than doubling ion extraction at high MS/MS frequencies. The new reflectron and detector improve resolving power compared to the previous model up to 80%, i.e. to 40,000 at m/z 1222. We analyzed the ion current from the inlet capillary and found very high transmission (>80%) up to the collision cell. Simulation and measurement indicated 60% transfer into the flight tube. We adapted MaxQuant for QTOF data, improving absolute average mass deviations to better than 1.45 ppm. More than 4,800 proteins can be identified in a single run of HeLa digest in a 90 min gradient. The workflow achieved high technical reproducibility (R2>0.99) and accurate fold change determination in spike-in experiments over three orders of magnitude in complex mixtures. Using label-free quantification we rapidly quantified haploid against diploid yeast and characterized overall proteome differences in mouse cell lines originated from different tissues. Finally, after high pH reversed-phase fractionation we identified 9,515 proteins in a triplicate measurement of HeLa peptide mixture and 11,257 proteins in cerebellum – the highest proteome coverage measured with a QTOF instrument so far.
2015-05-26 | PXD001592 | Pride
Project description:Plaques consisting of amyloid-β (Aβ) in the brain are characteristic for patients with Alzheimer´s disease. Aβ is enzymatically generated of the amyloid precursor protein. Furthermore, neprilysin, an Aβ-degrading enzyme, and its main activator the neuropeptide somatostatin (SST) are downregulated in Alzheimer´s disease. In the fusion protein SST-scFv8D3, SST is recombinantly fused to a blood-brain barrier transporter. The aim of this study was to study the effect of SST-scFv8D3 on the proteome of different brain regions in order to elucidate the anti-Alzheimer´s disease effects of this construct. SST-scFv8D3 and phosphate buffered saline (PBS) respectively were injected into transgenic mice of an Alzheimer´s disease model with the Swedish mutation (AβPP KM670/671NL) every 36 hours (three times in total). Homogenates of the hippocampus, the cerebellum and the rest of the cerebrum were lysed. Afterwards, filter aided tryptic digestion was performed. The resulting peptides were analyzed using LC-UDMSE. The data was searched against a randomized mouse database and label-free quantification analysis was done. In total 1869 proteins were found. Only the proteome of the hippocampus was affected by the treatment with SST-scFv8D3. 55 proteins were significantly up and 105 down regulated compared to the PBS treated mice. Among these were mitochondrial and synaptic proteins as well as some involved in neuron´s development indicating anti-Alzheimer disease effects of SST-scFv8D3 and supporting the future use of SST-scFv8D3 as a treatment option.
2021-06-28 | PXD022221 | Pride
Project description:Filter-aided sample preparation (FASP) is widely used in bottom-up proteomics for tryptic digestion. However, the sample recovery yield of this method is limited by the amount of starting material. While ~100 ng of digested protein sample is sufficient for thorough protein identification, proteomic information gets lost after digestion of samples with a protein content < 10 µg due to incomplete peptide recovery from the filter. By reducing the filter area as well as the volumes of all reagents, we developed and optimized a well-plate µFASP device and protocol that is suitable for ~1 µg protein sample. In 1 µg of HeLa digest, we identified 1295 ± 10 proteins with our method followed by analysis with liquid chromatography–mass spectrometry. In contrast, only 524 ± 5 proteins were identified with the standard FASP protocol while 1395 ± 4 proteins were identified in 20 µg after standard FASP as a benchmark. Furthermore, we used our method to conduct a combined peptidomic and proteo-mic study of single islets of Langerhans. Here, we separated neuropeptides from single islets as effluents from a ⌀ 1 mm molecular cut-off filter and digested the remaining on-filter proteins for bottom-up proteomic analysis. Our results indicate inter-islet heterogeneity for expression of proteins involved in glucose catabolism, pancreatic hormone processing and secreted peptide hormones. We consider our method to provide a useful tool for proteomic characterization of samples where biological material is scarce.
2022-04-01 | PXD029039 | Pride
Project description:Spatial tissue proteomics integrating whole-slide imaging, laser microdissection and ultrasensitive mass spectrometry is a powerful approach to link cellular phenotypes to functional proteome states in (patho)physiology. To be applicable to large patient cohorts and low sample input amounts, including single-cell applications, loss-minimized and streamlined end-to-end workflows are key. We here introduce an automated sample preparation protocol for laser microdissected samples utilizing the cellenONE® robotic system, which has the capacity to process 192 samples in three hours. Following laser microdissection collection directly into the proteoCHIP LF 48 or EVO 96 chip, our optimized protocol facilitates lysis, formalin de-crosslinking and tryptic digest of low-input archival tissue samples. The seamless integration with the Evosep ONE LC system by centrifugation allows ‘on-the-fly’ sample clean-up, particularly pertinent for laser microdissected workflows. We validate our method in human tonsil archival tissue, where we profile proteomes of spatially-defined B-cell, T-cell and epithelial microregions of 4,000 µm2 to a depth of ~2,000 proteins and with high cell type specificity. We finally provide detailed equipment templates and experimental guidelines for broad accessibility.
2024-03-22 | PXD047762 | Pride
Project description:The membrane glycoprotein M6a -together with proteolipid protein (PLP), DM20 and M6b- belongs to the tetraspan PLP family. M6a is a neuronal surface protein that promotes neuronal stem cell differentiation, migration, neurite and axonal outgrowth, filopodia/spine induction and synapse formation in primary neuronal cultures and non-neuronal cell lines. M6a has two extracellular loops (EC1 and EC2), four transmembrane domains, one intracellular loop and the N and C terminus facing the cell cytoplasm. However, the complete mechanism of action or proteins associated with it through its extracellular loops remained unknown. In previous work we provided strong evidence that M6a´s extracellular loops widely contribute to its function. To asses this question, we designed and purified a chimera protein (M6a-loops as a bait protein) which was subjected to co-immunoprecipitation (Co-Ip) with rat hippocampi homogenates followed by TMT-MS. The TMT-MS and data analysis was done by the Proteomics Core Facility from the European Molecular Biology Laboratory (EMBL, Heidelberg, Germany).
2020-06-29 | PXD017347 | Pride
Project description:The inflammatory functions of the cytokine tumor necrosis factor (TNF) rely on its ability to induce cytokine production and to induce cell death. Caspase dependent and independent pathways – apoptosis and necroptosis – respectively, regulate immunogenicity by the release of distinct sets of cellular proteins. To obtain an unbiased, systems-level understanding of this important process, we here applied mass spectrometry-based proteomics to dissect protein release during apoptosis and necroptosis. We report hundreds of proteins released from human myeloid cells in time-course experiments. Both cell death types induce receptor shedding, but only apoptotic cells released nucleosome components. Conversely, necroptotic cells release lysosomal components by activating lysosomal exocytosis at early stages of necroptosis- induced membrane permeabilisation and show reduced release of conventionally secreted cytokines.
2020-07-18 | PXD014966 | Pride
Project description:Here, we successfully used NO as the direct electron acceptor for the enrichment of a microbial community in a continuous bioreactor. The enrichment culture, mainly comprised of two new organisms from the Sterolibacteriaceae family, grew on NO reduction to N2 and formate oxidation, with virtually no accumulation of N2O. The microbial growth kinetics of the enrichment culture as well as its affinity for different N-oxides were determined. In parallel, using metagenomics, metatranscriptomics, and metaproteomics, the biochemical reactions underlying the growth of these microorganisms on NO were investigated. This study demonstrates that microorganisms thrive and can be enriched on NO, and presents new opportunities to study microbial growth on this highly energetic and climate-active molecule that may have been pivotal in the evolution of aerobic respiration.
2023-06-09 | PXD037586 | Pride
Project description:A time series of whole-genome transcription profiling of E. coli K-12 W3110 was performed to study the dynamic physiological response of E. coli K-12 W3110 to limited carbon conditions. cDNA from eight time intervals after glucose limitation, representing different stages of glucose depletion, along with cDNA of an unrestricted growth were hybridized to whole-genome microarrays of E. coli K-12. Glucose concentrations were gradually reduced in a fed-batch cultivation process with constant feeding rate in a bioreactor. Major focus was put on the dynamic changes in the transcription level of genes involved in the central carbon metabolism (glycolysis, pentose phosphate pathway, TCA cycle and glyoxylate shunt), high-affinity glucose transport systems, movement, growth, and stress response. In total, 40 microarrays were hybridized. The data were statistically analysed using the software R (R, 2005) and the Limma package included (Smyth, 2005). The data were normalized â??print-tip-loessâ??. â??Multiple hypothesis testingâ?? correction was performed with the method of Benjamini and Hochberg (Benjamini and Hochberg, 1995), which controls the â??false discovery rateâ?? (fdr). Genes were regarded as differentially expressed when the â??adjusted p valuesâ?? were < 0.05, therefore nominally controlling the expected fdr to less than 0.05. The reproducibility of dye swap experiments as well as biological replicates was estimated by calculating â??Pearsonâ??s correlation coefficientsâ??. A high correlation was observed for the dye swap pairs (with averaged Pearsonâ??s correlation coefficients of 0.89 ± 0.06 for the reference samples and of 0.87 ± 0.08 for the different points of time) as well as for biological replicates (with averaged Pearsonâ??s correlation coefficients of 0.86 ± 0.04 for the red channel (Cy5) and 0.80 ± 0.09 for the green channel (Cy3)). Benjamini, Y., and Hochberg, Y. (1995). Controlling the false discovery rate: A practical and powerful approach to multiple testing. J R Stat Soc B 57, 289-300. Smyth, G.K. (2005). Limma: Linear models for microarray data. In Bioinformatics and Computational Biology Solutions using R and Bioconductor, R. Gentleman, Carrey, V., Dudoit, S., Irizzary, I., Huber, W., ed. (New York, Springer), pp. 397-420. R (2005). R: A language and environment for statistical computing. (R Development Core Team). Supplementary files: Data_table_A: TabelleA_Signifikante.txt Description: Data table with coefficients of the linear model for every gene which was differentially expressed for at least one point in time according to the reference sample. The statistical analysis was performed as described in Series section (GSE10307) based on the normalized log2(test/ref) ratios in the Sample records' VALUE columns. Data_table_B1: Tabelle_B1; /E. coli/, unlimited growth via glucose limitation (glucose concentration < 0.05 gL-1) Data_table_B2: Tabelle_B2; /E. coli/, unlimited growth via glucose limitation, acetate concentration = 0.35 gL-1 Data_table_B3: Tabelle_B3; /E. coli/, unlimited growth via glucose limitation, 30 min after depletion of extracellular acetate Data_table_B4: Tabelle_B4; /E. coli/, unlimited growth via glucose limitation, 50 min after depletion of extracellular acetate Data_table_B5: Tabelle_B5; /E. coli/, unlimited growth via glucose limitation, 110 min after depletion of extracellular acetate Data_table_B6: Tabelle_B6; /E. coli/, unlimited growth via glucose limitation, 170 min after depletion of extracellular acetate Data_table_B7: Tabelle_B7; /E. coli/, unlimited growth via glucose limitation, 230 min after depletion of extracellular acetate Data_table_B8: Tabelle_B8; /E. coli/, unlimited growth via glucose limitation, 350 min after depletion of extracellular acetate Description: Data_tables_B1-B8 report statistical values (A: average intensity value (1/2log2(test*ref)); M: average expression value (log2(test/ref)), t and P.value ('adjusted P.value')) for every point in time (studied conditions are described above for the corresponding table). Every differentially expressed gene of the corresponding point in time according to the reference sample is listed. Genes were regarded as differentially expressed when their 'adjusted P.values' were < 0.05, therefore nominally controlling the expected fdr to less than 0.05. The data are based on maximal three biological replicates (three independent fed-batch cultivations). RT-PCR_results_7genes.txt and RT-PCR_protocol.PDF Keywords: Time course, stress response Hybridization involved the hybridization of a common reference (unlimited growth, batch phase) on all arrays together with each sample of interest. This allowed the direct comparison of the transcripts of a time series taken during fed-batch cultivation with the unlimited reference sample. All samples were taken in duplicates and the experiments were performed as dye swap experiments. The first four samples of the time series (T1 - T4) were taken from three individual fed-batch cultivations (three biological replicates), the subsequent four samples (T5 - T8) were taken only from two individual fed-batch cultivations. On each array, the genes were spotted in duplicates. Therefore, twelve replicates (duplicates on dye swaps for three cultivations) were analyzed for T1 â?? T4 and eight replicates (duplicates on dye swaps for two cultivations) were analyzed for T5 â?? T8.
2010-05-19 | E-GEOD-10307 | biostudies-arrayexpress
Project description:we aimed to provide an objective roadmap for histone sample preparation wherein the most notable findings and most important metrics were highlighted. This roadmap included a Histone Coverage Tool that allows researchers to make a snapshot of public or in-house data in search of a protocol that suits their specific needs. However, despite the fact that the coverage provided by a sample preparation workflow is pivotal to answer specific research questions, it should be noted that other metrics such as enzyme specificity and workflow variability can be equally important. Moreover, when it comes to histone sample preparation there are multiple additional challenges and pitfalls compared to regular bottom-up proteomics workflows. First there is the abundance and unequal distribution of arginine and lysine cleavage sites, which makes it virtually impossible to cover the entire histone code with a single bottom-up workflow. The most widely spread workaround for the abundance of cleavage sites is derivatization of lysine residues used to block tryptic digest and create longer peptides. However, this also introduces chemical noise, which obscures biological PTMs, while lowering sensitivity due to diluted signal. The most obvious alternative is an arginine specific digest. However, on the one hand, derivatization of lysine residues not only increases peptide length but also increases retention on the LC-system, while decreasing charge state distribution of the precursor ions. The latter presumably reduces instrumental variation of the workflow, which is remarkable since this source of variation is generally assumed to be constant in the field of LC-MS/MS based proteomics. On the other hand, lysine derivatization also increases the singly charged precursor fraction, which is usually not fragmented in regular data dependent acquisitions, thus increasing the risk of losing annotations. It is clear that not one histone sample preparation workflow will fit all research questions. However, we hope that this manuscript will guide researchers in making informed decisions about the many pitfalls and trade-offs involved in optimizing or assessing histone sample preparation workflows.
2021-04-27 | PXD024423 | Pride