Human Aqueous Humor Phospholipids in Control and Glaucomatous eyes
Ontology highlight
ABSTRACT:
ORGANISM(S): Human Homo Sapiens
TISSUE(S): Eye Tissue
DISEASE(S): Eye Disease
SUBMITTER: 
Sanjoy Bhattacharya
PROVIDER: ST000616 | MetabolomicsWorkbench | Wed May 31 00:00:00 BST 2017
REPOSITORIES: MetabolomicsWorkbench
Similar Datasets
Project description:We compared phospholipid (phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol) profiles of control and glaucomatous trabecular meshwork (TM) derived from human donors.Lipid extraction was performed using a modification of the Bligh and Dyer method, protein concentrations were determined using the Bradford method, and for select samples confirmed with densitometry of PHAST gels. Lipids were identified and subjected to ratiometric quantification using a TSQ quantum Access Max triple quadrupole mass spectrometer with precursor ion scan (PIS) or neutral ion loss scan (NLS), using appropriate class specific lipid standards.
2017-06-01 | ST000624 | MetabolomicsWorkbench
Project description:This study examined the profiles of sphin¬golipids and ceramides present in the aqueous humor (AH) of human control and POAG donors. Furthermore, we quantitatively compared distinct differences between glaucomatous and age-matched control eyes, identifying potential molecules for further experimentation to determine their biological role in modulating cell behavior. Lipids were identified and ratiometrically quantified in a two-step process using precursor ion scan (PIS) or neutral loss scan (NLS) with appropriate class-specific lipid standards on a TSQ Quantum Access Max mass spectrometer following established procedures. We identified several species of sphingomyelin, sphingoid base, sphingoid base-1-phosphate, and ceramides that were common between control and glaucomatous AQH. Some unique lipid species in these classes were also identified in controls but not in glaucoma and vice versa.
2017-05-26 | ST000612 | MetabolomicsWorkbench
Project description:Extracellular matrix (ECM) materials accumulate in the trabecular meshwork (TM) tissue of patients with glaucoma, which is associated with a decrease in aqueous humor outflow and therefore an increase in intraocular pressure. To explore a potential mechanism for ECM regulation in the TM, we purified extracellular vesicles (EVs) from conditioned media of differentiated TM cells in culture isolated from non-glaucomatous and glaucomatous human donor eyes. EVs were purified using the double cushion ultracentrifugation gradient method. Fractions containing EV markers CD9 and TSG101 were analyzed using nanoparticle tracking analysis to determine their size and concentration. We then determined their proteomic cargo by mass spectrometry and compared protein profiles of EVs between normal and glaucomatous TM cells using PANTHER. Key protein components from EV preparations were validated with Western blotting. Results showed changes in the percentage of ECM proteins associated with EVs from glaucomatous TM cells compared to non-glaucomatous TM cells (5.7% vs 13.1% respectively). Correspondingly, we found that two ECM-related cargo proteins found across all samples, fibronection and EDIL3 were significantly less abundant in glaucomatous EVs (<0.3 fold change across all groups) compared to non-glaucomatous EVs. Overall, these data establish that ECM materials are prominent cargo in EVs from TM cells, and their binding to EVs is diminished in glaucoma
2024-01-26 | PXD044913 | Pride
Project description:Isolated murine kidney glomeruli were lysed in buffer containing 8M urea, and proteins were reduced and alkylated. Proteins were digested with trypsin. After reverse-phase chomatrography, peptides were fractionated using strong cation exchange chromatography. Phosphopeptides were enriched using IMAC (FeNTA) columns. Peptides were analyzed by LC-MS/MS on a Q Exactive mass spectrometer or an LTQ Orbitrap XL mass spectrometer. Metadata for Orbitrap samples (MaxQuant Output files include „Orbi“) 1. General features – 1.1 Global descriptors a. Responsible person or role: Markus Rinschen. markus.rinschen@uk-koeln.de; tobias.lamkemeyer@uni-koeln.de b. Instrument manufacturer, model: LTQ-Orbitrap XL, Thermo Scientific c. customization 2. Ion sources – ESI fed by nLC 2 (Proxeon) 3. Post source component 3.1. Analyser: MS1 survey scan in an Orbitrap and MS2 analysed in Linear Trap. 3.2. Activation/Dissociation: CID 4. Spectrum and peak list generation and annotation 4.1. Data acquisition: MaxQuant v. 1.3.05 Top one method with a cycle of one full MS1 scan in the Orbitrap, followed by the fragmentation with dynamic exclusion window of 60 seconds and followed by the acquisition of 5 product ion scans generated in the LTQ analyser, and detected in the LTQ. Unselected fragmentation. For further details, see .raw files. URL of file: Filename 4.2. Data analysis: MaxQuant v 1.3.05 Parameters used in the generation of peak lists or processed spectra: see annotation in “parameters” file. The mouse uniprot reference database “complete proteome” obtained on January 2nd, 2013 was used. Metadata for QExactive samples (Max Quant output files include „QE“) 1. General features – 1.1 Global descriptors a. Responsible person or role: Markus Rinschen. markus.rinschen@uk-koeln.de; Marcus Krüger marcus.krueger@mpi-bn.mpg.de b. Instrument manufacturer, model: Q Exactive Thermo Scientific c. customization 2. Ion sources – ESI fed by nLC 1000 (Proxeon) 3. Post source component 3.1. Analyser: MS1 survey scan in an Orbitrap and MS2 analysed in Orbitrap/HCD cell 3.2. Activation/Dissociation: HCD 4. Spectrum and peak list generation and annotation 4.1. Data acquisition: MaxQuant v. 1.3.05 Top one method with a cycle of one full MS1 scan in the Orbitrap, followed by the fragmentation with dynamic exclusion window of 20 seconds in the HCD cell and followed by the acquisition of 10 product ion scans generated in the LTQ analyser, and detected in the LTQ. Unselected fragmentation. For further details, see .raw files. URL of file: Filename 4.2. Data analysis: MaxQuant v 1.3.05 Parameters used in the generation of peak lists or processed spectra: see annotation in “parameters” file. The mouse uniprot reference database “complete proteome” obtained on January 2nd, 2013 was used.
2016-05-09 | PXD000266 | Pride
Project description:Trabecular meshwork cells in eyes with glaucoma aquire mesenchymal phenotypes. The types of microRNAs in exosomes may differ between static and glaucomatous status and their effects on aqueous humor regulation are still uknown. We used microarrays to identify the differential microRNA expression related to interaction between trabecular meshwork cells and Schlemm's canal endothelial cells.
2021-11-17 | GSE175464 | GEO
Project description:TGF-beta levels are known to increase in the aqueous humor of eye cells in patients with glaucoma. Increase TGF-beta is assumed to have a biochemical impact on the trabecular meshwork, and an increase in extracellular matrix formation, which may be responsible for decrease outflow facility of the eye. This may increase extracellular pressure, causing glaucoma. TGF-beta 1 may be the cause of abnormal accumulation of extracellular matrices in trabecular meshwork of eyes with primary open angle glaucoma. Transforming growth factor (TGF)-beta2 regulates the expression of proteoglycans in aqueous humor from human glaucomatous eyes. To identify gene expression changes as a result of TGF-beta1 and 2 treatment of human trabecular meshwork cells. We expect to see a change in expression of the proteoglycans in HTM cells as a response to TGF-beta treatment. Human Trabecular Meswork cells in the eye were bathed by aqueous humor. TM cells were removed from individuals with the following ages: 16,66,67,73, and 76. Each individual was treated with EtOH (control), TGF-beta1, or TGF-beta2. Total RNA from each individual was pooled for each chip. Technical replicates were created for each treatment type, for a total of 6 chips.
2008-06-15 | E-GEOD-7144 | biostudies-arrayexpress
Project description:Interventions: Gold Standard:pathological diagnosis ;Index test:Volatile organic compounds detected by gas chromatography-mass spectrometer and gas chromatography-ion migration spectrometry
Primary outcome(s): Volatile organic compounds;sensitivity;specificity
Study Design: Diagnostic test for accuracy
| 89221 | ecrin-mdr-crc
Project description:Untargeted metabolomics aims at obtaining quantitative information on the highest possible number of low-molecular biomolecules present in a biological sample. Rather small changes in mass spectrometric spectrum acquisition parameters may have a significant influence on the detectabilities of metabolites in untargeted global-scale studies by means of high-performance liquid chromatography-mass spectrometry (HPLC-MS). Employing whole cell lysates of human renal proximal tubule cells, we present a systematic global-scale study of the influence of mass spectrometric scan parameters on the number and intensity of metabolites detectable in whole cell lysates. Ion transmission and ion collection efficiencies in an Orbitrap-based mass spectrometer generally depend on the m/z range scanned, which, ideally, requires different instrument settings for the respective mass ranges investigated. Therefore, we split a full scan range of m/z 50-1000 relevant for metabolites into two separate segments (m/z 50-200 and m/z 200-1000), allowing an independent tuning of the ion transmission parameters for both mass ranges. Three different implementations, involving either scanning from m/z 50-1000 in a single scan, or scanning from m/z 50-200 and from m/z 200-1000 in two alternating scans, or performing two separate HPLC-MS runs with m/z 50-200 and m/z 200-1000 scan ranges were critically assessed. The most efficient approach in terms of feature number, which forms the basis for statistical analysis, identification, and for generating biological hypotheses, was the separate analysis of two different mass ranges. This lead to an increase in the number of detectable metabolite features, especially in the higher mass range (m/z greater than 400), by 2.5 (negative mode) to 6-fold (positive mode) as compared to analysis involving a single scan range. Application of the method to metabolite extracts prepared from whole cell lysates of human renal proximal tubule epithelial cells initially yielded up to 13,000 – 69,000 detectable features, which were subjected to rigorous background filtering and quality control in order to obtain reliable metabolite features for subsequent differential quantification.
2016-06-09 | MTBLS233 | MetaboLights
Project description:Methylation of lysine 4 at histone H3 (H3K4) at promoters is tightly linked to transcriptional regulation in human cells. At least six different COMPASS-like multi-subunit (SET1/MLL) complexes have been described that contain methyltransferase activity towards H3K4, but a comprehensive and quantitative analysis of these SET1/MLL complexes is lacking. We applied label-free quantitative mass spectrometry to determine the subunit composition and stoichiometry of the human SET1/MLL complexes. Peptides were applied to online nanoLC-MS/MS, using a 120 min acetonitrile gradient (5.6 - 76%). Mass spectra were recorded on an LTQ-Orbitrap-Velos mass spectrometer (Thermo) selecting the 15 most intense precursor ions of every full scan for fragmentation. Raw data were analyzed using MaxQuant 1.3.0.5, with label-free quantification (LFQ), match between runs (between triplicates) and the iBAQ algorithm enabled. MaxQuant default settings were used for peptide identification; Enzyme: Trypsin/P, MS tolerance (FTMS): 6 ppm, max missed cleavages: 2, max charge: 7, MS/MS tolerance (ITMS): 0.5 Da, Peptide FDR: 0.01 and Protein FDR: 0.01. Normalized mass spectrometric intensities (LFQ intensities) were compared between the GFP-tagged and control sample, using an adapted permutation-based false discovery rate (FDR) t-test in Perseus (MaxQuant package).
2013-09-13 | PXD000172 | Pride