Molecular Response of Retinal Pigment Epithelial Cells to Oxidized Lipoproteins: Global and Targeted Studies
type: CreativeWork
description: Global-scale examinations of biological systems at the molecular level complement targeted approaches to scientific inquiry that focus on specific subsets of biomolecules, or on a single molecule of interest. In this dissertation, we utilized both the discovery-based approach to evaluate the proteomics workflows centered around mass spectrometry as the key technology, and the targeted approach to examine the molecular response of RPE due to oxidized lipoproteins (oxLDL) treatments. A crucial aspect in proteomics studies is the design of bioanalytical strategies that maximize coverage of the complex repertoire of a proteome. A comprehensive, unbiased examination of the proteome represents a powerful approach toward system-level insights into disease mechanisms. We evaluated the performance of bioanalytical platforms for profiling of the proteome in a biological system. We applied a discovery-based approach to evaluate the global transcriptome and proteome changes due to oxLDL treatment in ARPE-19 cells. We studied the role of scavenger receptors CD36 and CD5L/AIM in ARPE-19 cells when induced with oxLDL. We compared three different multidimensional proteome fractionation platforms: polymeric reversed-phase liquid chromatography at high pH (PLRP), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and isoelectric focusing (IEF) separations. We applied a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) in a data-dependent setting and used bioinformatics for protein identification. The three platforms identified a total of 1043 proteins altogether. Among the three bioanalytical strategies, SDS-PAGE followed by LC-MS/MS provided the best coverage. We also evaluated another bioanalytical platform which consists of a highresolution mass spectrometer combined with nano-UPLC in a data-independent setting without pre-fractionation for oxLDL mediated proteome alteration in ARPE-19 cells. This platform outperformed the SDS-PAGE based analy…
abstract: Global-scale examinations of biological systems at the molecular level complement targeted approaches to scientific inquiry that focus on specific subsets of biomolecules, or on a single molecule of interest. In this dissertation, we utilized both the discovery-based approach to evaluate the proteomics workflows centered around mass spectrometry as the key technology, and the targeted approach to examine the molecular response of RPE due to oxidized lipoproteins (oxLDL) treatments. A crucial aspect in proteomics studies is the design of bioanalytical strategies that maximize coverage of the complex repertoire of a proteome. A comprehensive, unbiased examination of the proteome represents a powerful approach toward system-level insights into disease mechanisms. We evaluated the performance of bioanalytical platforms for profiling of the proteome in a biological system. We applied a discovery-based approach to evaluate the global transcriptome and proteome changes due to oxLDL treatment in ARPE-19 cells. We studied the role of scavenger receptors CD36 and CD5L/AIM in ARPE-19 cells when induced with oxLDL. We compared three different multidimensional proteome fractionation platforms: polymeric reversed-phase liquid chromatography at high pH (PLRP), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and isoelectric focusing (IEF) separations. We applied a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) in a data-dependent setting and used bioinformatics for protein identification. The three platforms identified a total of 1043 proteins altogether. Among the three bioanalytical strategies, SDS-PAGE followed by LC-MS/MS provided the best coverage. We also evaluated another bioanalytical platform which consists of a highresolution mass spectrometer combined with nano-UPLC in a data-independent setting without pre-fractionation for oxLDL mediated proteome alteration in ARPE-19 cells. This platform outperformed the SDS-PAGE based analy…
author: Koirala, Diwa
keywords: Age Related Macular Degeneration, Bioanalytics, Mass Spectrometry, Proteomics, Retinal Pigment Epithelium, Medicine and Health Sciences
date published: 2018-12-01T08:00:00Z
date modified: 2026-07-24T05:00:33Z
provider: University of Tennessee Health Science Center
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Tennessee Health Science Center