Sphingolipid Analysis of Human Aqueous Humor in Glaucomatous and Control eyes (part I)
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
TISSUE(S): Eye Tissue
DISEASE(S): Eye Disease
PROVIDER: ST000612 | MetabolomicsWorkbench |
REPOSITORIES: MetabolomicsWorkbench
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Project description:PurposeHuman aqueous humor (hAH) provides nutrition and immunity within the anterior chamber of the eye. Characterization of the protein composition of hAH will identify molecules involved in maintaining a homeostatic environment for anterior segment tissues. The present study was conducted to analyze the proteome of hAH.MethodshAH samples obtained during elective cataract surgery were divided into three matched groups and immunodepleted of albumin, IgG, IgA, haploglobin, antitrypsin, and transferrin. Reduced and denatured proteins (20 μg) from each group were separated by gel electrophoresis. Thirty-three gel slices were excised from each of three gel lanes (n = 99), digested with trypsin, and subjected to nanoflow liquid chromatography electrospray ionization tandem mass spectrometry (nano-LC-ESI-MS/MS). The protein component of hAH was also analyzed by antibody-based protein arrays, and selected proteins were quantified.ResultsA total of 676 proteins were identified in hAH. Of the 355 proteins identified by nano-LC-ESI-MS/MS, 206 were found in all three groups. Most of the proteins identified by nano-LC-ESI-MS/MS had catalytic, enzymatic, and structural properties. Using antibody-based protein arrays, 328 cytokines, chemokines, and receptors were identified. Most of the quantified proteins had concentrations that ranged between 0.1 and 2.5 ng/mL. Ten proteins were identified by both nano-LC-ESI-MS/MS and antibody protein arrays.ConclusionsProteomic analysis of hAH identified 676 nonredundant proteins. More than 80% of these proteins are novel identifications. The elucidation of the aqueous proteome will establish a foundation for protein function analysis and identification of differentially expressed markers associated with diseases of the anterior segment.
| S-EPMC3066620 | biostudies-literature
Project description: Not available
| ST000616 | MetabolomicsWorkbench
Project description:To compare phospholipid (phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and phosphatidylinositol) profiles of human control and glaucomatous aqueous humor (AQH). AQH samples were procured during surgery from human POAG and control subjects (n = 15 each). Samples were used following institutional review board approved protocols and adhering to the tenets of the Declaration of Helsinki. Lipid extraction was performed using a modification of the Bligh and Dyer method, protein concentrations were determined using the Bradford's method, and select samples were confirmed with Densitometry of PHAST gels. Lipids were identified and subjected to ratiometric quantification using a TSQ Quantum Access Max triple quadrupole mass spectrometer utilizing precursor ion scan (PIS) or neutral ion loss scan (NLS) using appropriate class specific lipid standards in a two step quantification process. The comparative profiles of phosphatidylcholines, phosphatidylserines, phosphatidylethanolamines and phosphatidylinositols between control and glaucomatous AQH showed several species common between them. A number of unique lipids in all four phospholipid classes were also identified in control eyes that were absent in glaucomatous eyes and vice versa. A number of phospholipids were found to be uniquely present in control, but absent in glaucomatous AQH and vice versa. Compared with a previous study of control and POAG red blood cells, a number of these phospholipids are absent locally (AQH).
| S-EPMC3995849 | biostudies-literature
Project description: Not available
| ST000620 | MetabolomicsWorkbench
Project description:Aqueous humor (AH) supports avascular tissues in the anterior segment of the eye, maintains intraocular pressure, and potentially influences the pathogenesis of ocular diseases. Nevertheless, the AH proteome is still poorly defined despite several previous efforts, which were hindered by interfering high abundance proteins, inadequate animal models, and limited proteomic technologies. To facilitate future investigations into AH function, the AH proteome was extensively characterized using an advanced proteomic approach. Samples from patients undergoing cataract surgery were pooled and depleted of interfering abundant proteins and thereby divided into two fractions: albumin-bound and albumin-depleted. Multidimensional Protein Identification Technology (MudPIT) was utilized for each fraction; this incorporates strong cation exchange chromatography to reduce sample complexity before reversed-phase liquid chromatography and tandem mass spectrometric analysis. Twelve proteins had multi-peptide, high confidence identifications in the albumin-bound fraction and 50 proteins had multi-peptide, high confidence identifications in the albumin-depleted fraction. Gene ontological analyses were performed to determine which cellular components and functions were enriched. Many proteins were previously identified in the AH and for several their potential role in the AH has been investigated; however, the majority of identified proteins were novel and only speculative roles can be suggested. The AH was abundant in anti-oxidant and immunoregulatory proteins as well as anti-angiogenic proteins, which may be involved in maintaining the avascular tissues. This is the first known report to extensively characterize and describe the human AH proteome and lays the foundation for future work regarding its function in homeostatic and pathologic states.
| S-EPMC2793904 | biostudies-literature
Project description: Not available
| S-EPMC5522757 | biostudies-literature
Project description: Not available
| ST000613 | MetabolomicsWorkbench
Project description:A miRNA PCR array comprising 84 miRNAs was used to analyze the AH (glaucoma, n=3; control, n=3) and LC samples (glaucoma, n=3; control, n=4). Expression levels of 19 and 3 miRNAs were significantly upregulated in the AH and LC samples of the glaucoma group, respectively (p < 0.05).
2024-02-16 | E-MTAB-13771 | biostudies-arrayexpress
Project description:PurposeAqueous humor is intimately related to the cells of the anterior and posterior chambers, which affect its composition. Aqueous analysis provides useful information regarding physiological and pathophysiological processes in the eye. Human aqueous samples are typically less than 100 microl, limiting the usefulness of the analysis with traditional Enzyme-Linked immunoSorbant Assay (ELISA) techniques. The specific aim of this study was to investigate if whether large numbers of analytes can be identified in clinically available samples of aqueous humor and to document the detectability of certain biomarkers in the aqueous.MethodsWe used a technology developed by Luminex xMAP to analyze hundreds of analytes in a small sample. Aqueous from eight normal and two diabetic patients was analyzed.ResultsOf the 90 analytes evaluated, 52 (57%) were detectable in the normal aqueous. To place these results in biological context, we analyzed the list of expressed analytes using the MetaCore database. The functional pathways, networks, biological processes, and disease processes that these analytes represented were identified. Several ocular pathology-related processes were represented in the aqueous. The detected analytes represented biomarkers of several relevant disease processes including vascular diseases, arteriosclerosis, ischemia, necrosis, and inflammation. To provide the proof of principle that the aqueous profile could offer useful information about the pathophysiological processes, we analyzed two aqueous samples from diabetic patients. These limited samples showed the differences between normal and diabetic samples, including those relevant to diabetic retinopathy such as vascular endothelial growth factor (VEGF), C reactive protein, glutathione, and cytokines. Several biomarker groups for disease processes relevant to diabetes were perturbed.ConclusionsThese results demonstrate that multiplex analysis of the aqueous can be a useful tool in screening for any pathophysiological changes of the ocular environment. Moreover, ocular pathology/pathophysiology-specific Multi-analyte profiles MAPs can be developed and used to analyze the aqueous.
| S-EPMC2622713 | biostudies-literature
Project description:In order to determine the possible aqueous humor (AH) proteins involved in diabetic nephropathy (DN) progression, we performed gel electrophoresis-liquid chromatography-tandem mass spectrometry protein profiling of AH samples from 5 patients with proliferative diabetic retinopathy (PDR) combined DN and 5 patients with PDR. Function enrichment analyses were carried out after the identification of differentially expressed proteins (DEPs). Protein-protein interaction networks were then built and the Search Tool for the Retrieval of Interacting Genes database and CytoNCA plugin in Cytoscape were utilized for module analysis. Ingenuity Pathway Analysis (IPA) was used to analyze disease and biological function, Tox function enrichment and upstream regulatory molecules/networks. Fifty-four DEPs were finally confirmed, whose enriched functions and pathways covered cell adhesion, extracellular exosome, complement activation, complement and coagulation cascades, etc. Nine hub genes were identified, including NCAM1, PLG, APOH, C3, PSAP, RBP4, CDH2, NUCB1, and GNS. IPA showed that C3 and PLG are involved in renal and urological system abnormalities. Conclusively, DEPs and hub proteins confirmed in this exploratory AH proteomic analysis may help us gain a deeper understanding of the molecular mechanisms involved in DN progression, providing novel candidate biomarkers for the early detection for diagnosis of DN.
| S-EPMC9537621 | biostudies-literature