Cytoplasmic proteomic analysis of glioblastoma cells using a label free quantitative method
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Exploris 480
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Helene Perreault
PROVIDER: MSV000093417 | MassIVE | Fri Nov 17 08:21:00 GMT 2023
REPOSITORIES: MassIVE
Similar Datasets
Project description:Knockdown of SUPT6H (aka Spt6) using siRNA against Spt6 compared to treatement with scrambled CTRL siRNA in patient derived glioblastoma cell line
2020-07-17 | GSE125621 | GEO
Project description:Information on the composition of protein complexes can accelerate mechanistic analyses of cellular systems. Protein complex composition identifies genes that function together and provides clues about regulation within and between cellular pathways. Cytosolic protein complexes control metabolic flux, signal transduction, protein abundance, and the activities of cytoskeletal and endomembrane systems. It has been estimated that one third of all cytosolic proteins in leaves exist in an oligomeric state, yet the composition of nearly all remain unknown. Subunits of stable protein complexes copurify, and combinations of mass-spectrometry-based protein correlation profiling and bioinformatic analyses have been used to predict protein complex subunits. Because of uncertainty regarding the power or availability of bioinformatic data to inform protein complex predictions across diverse species, it would be highly advantageous to predict composition based on elution profile data alone. Here we describe a mass spectrometry-based protein correlation profiling approach to predict the composition of hundreds of protein complexes based on biochemical data. Extracts were obtained from an intact organ and separated in parallel by size and charge under nondenaturing conditions. More than 1000 proteins with reproducible elution profiles across all replicates were subjected to clustering analyses. The resulting dendrograms were used to predict the composition of known and novel protein complexes, including many that are likely to assemble through self-interaction. An array of validation experiments demonstrated that this new method can drive protein complex discovery, guide hypothesis testing, and enable systems-level analyses of protein complex dynamics in any organism with a sequenced genome.
| S-EPMC6683005 | biostudies-literature
Project description:Successful quantitative mass spectrometry (MS) requires strategies to link the mass spectrometer response to the analyte abundance, with the response being dependent on more factors than just analyte abundance. Label-dependent strategies rely on the incorporation of an isotopically labeled internal standard into the sample. Current label-free strategies (performed without internal standards) are useful for analyzing samples that are unsuitable for isotopic labeling but are less accurate. Here we describe a label-free technique applicable to analysis of products from related genes (isotypes). This approach enables the invariant tryptic peptide sequences within the family to serve as "built-in" internal standards and the isotype-specific peptide sequences to report the amount of the various isotypes. A process of elimination segregates reliably trypsin-released standard and reporter peptides from unreliably released peptides. The specific MS response factors for these reporter and standard peptides can be determined using synthetic peptides. Analysis of HeLa tubulin digests revealed peptides from betaI-, betaII-, betaIII-, betaIVb-, and betaV-tubulin, eight of which were suitable; along with five standard peptides for quantification of the beta-tubulin isotypes. To show the utility of this method, we determined that betaI-tubulin represented 77% and betaIII-tubulin represented 3.2% of the total HeLa beta-tubulin.
| S-EPMC3711517 | biostudies-literature
Project description:Label-free quantification is a powerful method for studying cellular protein phosphorylation dynamics. However, whether current data normalization methods achieve sufficient accuracy has not been examined systematically. Here, we demonstrate that a large uni-directional shift in the phosphopeptide abundance distribution is problematic for global median centering and quantile-based normalizations and may mislead the biological conclusion from unlabeled phosphoproteome data. Instead, we present a novel normalization strategy, named pairwise normalization, which is based on adjusting phosphopeptide abundances measured before and after the enrichment. The superior performance of pairwise normalization was validated by statistical methods, western blotting analysis, and by bioinformatics analysis. In addition, we demonstrate that the choice of normalization method influences the downstream analyses of the data and perceived pathway activities. Furthermore, we demonstrate that the developed normalization method, combined with pathway analysis algorithms, revealed a novel biological synergism between Ras signalling and PP2A inhibition by CIP2A.
2015-08-24 | PXD001374 | Pride
Project description:Glucose uptake assays commonly rely on the isotope-labeled sugar, which is associated with radioactive waste and exposure of the experimenter to radiation. Here, we show that the rapid decrease of the cytosolic pH after a glucose pulse to starved Saccharomyces cerevisiae cells is dependent on the rate of sugar uptake and can be used to determine the kinetic parameters of sugar transporters. The pH-sensitive green fluorescent protein variant pHluorin is employed as a genetically encoded biosensor to measure the rate of acidification as a proxy of transport velocity in real time. The measurements are performed in the hexose transporter-deficient (hxt0) strain EBY.VW4000 that has been previously used to characterize a plethora of sugar transporters from various organisms. Therefore, this method provides an isotope-free, fluorometric approach for kinetic characterization of hexose transporters in a well-established yeast expression system.
| S-EPMC7811510 | biostudies-literature
Project description:Here we report a versatile method by which the interaction between a protein and a small molecule, and the disruption of that interaction by competition with other small molecules, can be monitored electrochemically directly in complex sample matrices.
| S-EPMC2861035 | biostudies-literature
Project description:Temozolomide (TMZ) is considered a first line chemotherapy drug for glioblastoma (GBM). Unfortunately, the GBM without methylation of O6-methylguanine-DNA methyltransferase (MGMT), accounting for about 70% of all GBM, shows an inherent resistance to TMZ treatment. Aberrant accumulation of neutral lipids, primarily triglycerides (TGs) and cholesteryl esters (CEs), in lipid droplets (LDs) has been recognized as metabolic vulnerability for GBM therapy. However, it is not known whether MGMT methylation affects lipid accumulation in GBM. Herein, we employed label-free Raman spectromicroscopy, which integrated stimulated Raman scattering (SRS) microscopy and confocal Raman spectroscopy, to quantitatively analyze both the amount and composition of intracellular LDs in intact GBM tissues obtained from patients who had undergone resection surgery. Our results showed significant reductions in both the LD amount and the CE percentage in MGMT unmethylated GBMs (MGMT methylation < 15%) compared to MGMT methylated ones (MGMT methylation ≥ 15%). Due to a big variation of lipid accumulation in the MGMT methylated GBMs, these patients were further divided into hypermethylated group (MGMT methylation ≥ 50%) and intermediate-methylated group (MGMT methylation 15∼50%), according to the significantly different median survival rates of these two groups. Remarkable differences in LD amount, CE percentage, and also lipid saturation degree were found between the hypermethylated group and the other two groups, but not between the unmethylated and intermediate-methylated groups. To elucidate the possible underlying mechanism, we analyzed the differential expression of lipid metabolism-related genes in GBM with different levels of MGMT methylation using The Cancer Genome Atlas Program (TCGA) dataset. It was shown that the genes related to lipid oxidation and lipid efflux were upregulated, and the genes related to lipid synthesis were downregulated in unmethylated group. These findings unravel the relationship between MGMT methylation and lipid accumulation in GBM, which may offer new opportunities for the diagnosis and treatment of TMZ-resistant GBM.
| S-EPMC10413324 | biostudies-literature
Project description:BackgroundCurrent mesoscale 3D imaging techniques are limited to transparent or cleared samples or require the use of X-rays. This is a severe limitation for many research areas, as the 3D color surface morphology of opaque samples-for example, intact adult Drosophila, Xenopus embryos, and other non-transparent samples-cannot be assessed. We have developed "ALMOST," a novel optical method for 3D surface imaging of reflective opaque objects utilizing an optical projection tomography device in combination with oblique illumination and optical filters.ResultsAs well as demonstrating image formation, we provide background information and explain the reconstruction-and consequent rendering-using a standard filtered back projection algorithm and 3D software. We expanded our approach to fluorescence and multi-channel spectral imaging, validating our results with micro-computed tomography. Different biological and inorganic test samples were used to highlight the versatility of our approach. To further demonstrate the applicability of ALMOST, we explored the muscle-induced form change of the Drosophila larva, imaged adult Drosophila, dynamically visualized the closure of neural folds during neurulation of live Xenopus embryos, and showed the complementarity of our approach by comparison with transmitted light and fluorescence OPT imaging of a Xenopus tadpole.ConclusionThus, our new modality for spectral/color, macro/mesoscopic 3D imaging can be applied to a variety of model organisms and enables the longitudinal surface dynamics during development to be revealed.
| S-EPMC6323867 | biostudies-literature
Project description:Functional bone marrow studies have focused primarily on hematopoietic progenitors, leaving limited knowledge about other fragile populations, such as bone marrow adipocytes (BMAds) and megakaryocytes. The isolation of these cells is challenging due to rupture susceptibility and large size. We introduce here a label-free cytometry microsystem, MarrowCellDLD, based on deterministic lateral displacement. MarrowCellDLD enables the isolation of large, fragile BM-derived cells based on intrinsic size properties while preserving their viability and functionality. Bone marrow adipocytes, obtained from mouse and human stromal line differentiation, as well as megakaryocytes, from primary human CD34+ hematopoietic stem and progenitor cells, were used for validation. Precise micrometer-range separation cutoffs were adapted for each cell type. Cells were sorted directly in culture media, without pre-labeling steps, and with real-time imaging for quality control. At least 106 cells were retrieved intact per sorting round. Our method outperformed two FACS instruments in purity and yield, particularly for large cell size fractions. MarrowCellDLD represents a non-destructive sorting tool for large, fragile BM-derived cells, facilitating the separation of pure populations of BMAds and megakaryocytes to further investigate their physiological and pathological roles.
| S-EPMC10725893 | biostudies-literature
Project description:A new label-free molecular probe for luminescent nucleotide detection in neutral aqueous solution is presented. Phosphate-containing molecules, such as nucleotides possess vital role in cell metabolism, energy economy, and various signaling processes. Thus, the monitoring of nucleotide concentration and nucleotide related enzymatic reactions is of high importance. Two component lanthanide complex formed from Tb(III) ion carrier and light harvesting antenna, readily distinguishes nucleotides containing different number of phosphates and enable direct detection of enzymatic reactions converting nucleotide triphosphate (NTP) to nucleotide di/monophosphate or the opposite. Developed sensor enables the detection of enzymatic activity with a low nanomolar sensitivity, as highlighted with K-Ras and apyrase enzymes in their hydrolysis assays performed in a high throughput screening compatible 384-well plate format.
| S-EPMC6264117 | biostudies-literature