Subcellular Fractionation Methods for the Quantification of Endogenous Protein in Human Liver Using Targeted Proteomics
type: CreativeWork
description: Ultra-performance Liquid chromatography-multiple reaction monitoring (UPLC-MRM) is a versatile mass spectrometry method with many applications in drug development. Due to its high selectivity, fast analysis time and high sensitivity, UPLC-MRM is widely used in sample bioanalysis for pharmacokinetics. Recently, interest into human endogenous protein quantification using targeted quantitative proteomics has increased. These quantification measurements are useful in many aspects of drug discovery and development, including protein biomarker monitoring, the development of a more accurate physiological-based pharmacokinetic (PBPK) model, and the support for expression-activity correlations in clinical pharmacology studies. Different from traditional xenobiotic bioanalysis, human endogenous protein quantifications using targeted proteomics face many challenges, such as high interference from matrix background and the low abundance of analyte itself. Therefore, it is important to incorporate enrichment during sample preparation in endogenous protein quantifications. Traditionally, solid phase extraction, protein precipitation, and filter-aided sample preparation could help clean up the target analyte. However, more targeted enrichment methods are needed to quantify low abundant endogenous proteins. One of these methods is immunocapture, which utilizes a specific antibody to enrich either the protein itself or digested tryptic peptides from the complex biological matrices. Because highly specific antibodies are required, this method cannot be extended to the quantification of every endogenous protein. For tissue protein analysis, a different enrichment strategy is often employed, i.e., subcellular fractionation. This method allows the enrichment of targeted proteins based on their subcellular location by isolating the corresponding organelles. In this dissertation, subcellular fractionation methods were incorporated into the conventional UPLC-MRM targeted proteomics workflo…
abstract: Ultra-performance Liquid chromatography-multiple reaction monitoring (UPLC-MRM) is a versatile mass spectrometry method with many applications in drug development. Due to its high selectivity, fast analysis time and high sensitivity, UPLC-MRM is widely used in sample bioanalysis for pharmacokinetics. Recently, interest into human endogenous protein quantification using targeted quantitative proteomics has increased. These quantification measurements are useful in many aspects of drug discovery and development, including protein biomarker monitoring, the development of a more accurate physiological-based pharmacokinetic (PBPK) model, and the support for expression-activity correlations in clinical pharmacology studies. Different from traditional xenobiotic bioanalysis, human endogenous protein quantifications using targeted proteomics face many challenges, such as high interference from matrix background and the low abundance of analyte itself. Therefore, it is important to incorporate enrichment during sample preparation in endogenous protein quantifications. Traditionally, solid phase extraction, protein precipitation, and filter-aided sample preparation could help clean up the target analyte. However, more targeted enrichment methods are needed to quantify low abundant endogenous proteins. One of these methods is immunocapture, which utilizes a specific antibody to enrich either the protein itself or digested tryptic peptides from the complex biological matrices. Because highly specific antibodies are required, this method cannot be extended to the quantification of every endogenous protein. For tissue protein analysis, a different enrichment strategy is often employed, i.e., subcellular fractionation. This method allows the enrichment of targeted proteins based on their subcellular location by isolating the corresponding organelles. In this dissertation, subcellular fractionation methods were incorporated into the conventional UPLC-MRM targeted proteomics workflo…
author: Qiu, Xiazi
language: en
keywords: Pharmaceutical sciences, bioanalysis, protein quantification, Subcellular Fractionation, Targeted proteomics, UPLC-MRM
date published: 2020-12-31
date modified: 2026-07-24T02:47:27Z
provider: University of Kansas
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Kansas

PropertyValue
property ID: Handle
value: https://hdl.handle.net/1808/37157