{"id":{"repo_id":"ku","oai_identifier":"oai:kuscholarworks.ku.edu:1808/37157"},"canonical_url":"https://search.dev.ndltd.org/etd/ku/oai:kuscholarworks.ku.edu:1808/37157","repository":{"repo_id":"ku","name":"University of Kansas","base_url":"https://kuscholarworks.ku.edu/server/oai/request"},"display":{"title":"Subcellular Fractionation Methods for the Quantification of Endogenous Protein in Human Liver Using Targeted Proteomics","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 workflow as sample enrichment tools to demonstrate their importance in quantifying human liver endogenous proteins. First, human liver neonatal Fc receptor and beta 2-microglobulin were enriched in a post-nuclear supernatant, and a UPLC-MRM method was developed and validated to quantify these two proteins. Our quantitative results and determination of interindividual variability will aid the future development of more accurate PBPK models for immunoglobulin G- or albumin- based biotherapeutics. Second, UPLC-MRM methods were developed for a panel of organelle marker proteins to accelerate the quality assessment of organelles isolated from a sucrose gradient. Lastly, commercially available organelle isolation kits were assessed for their compatibilities and enrichment efficiencies in UPLC-MRM workflows for the targeted quantification of human hepatic drug transporters and xenobiotic nuclear receptors. In summary, this dissertation demonstrates the suitability of subcellular fractionation as a sample enrichment tool in the quantification of human liver endogenous proteins using MRM-based targeted proteomics. These methods expand the current toolbox in target enrichment from tissue samples, and the results will aid monitoring the distribution of future tissue biomarkers and tissue biotherapeutics, and can be used in the development of PBPK models to predict exposure and therapeutic effects of small molecule drugs, as well as biotherapeutics.","abstract_html":"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 workflow as sample enrichment tools to demonstrate their importance in quantifying human liver endogenous proteins. First, human liver neonatal Fc receptor and beta 2-microglobulin were enriched in a post-nuclear supernatant, and a UPLC-MRM method was developed and validated to quantify these two proteins. Our quantitative results and determination of interindividual variability will aid the future development of more accurate PBPK models for immunoglobulin G- or albumin- based biotherapeutics. Second, UPLC-MRM methods were developed for a panel of organelle marker proteins to accelerate the quality assessment of organelles isolated from a sucrose gradient. Lastly, commercially available organelle isolation kits were assessed for their compatibilities and enrichment efficiencies in UPLC-MRM workflows for the targeted quantification of human hepatic drug transporters and xenobiotic nuclear receptors. In summary, this dissertation demonstrates the suitability of subcellular fractionation as a sample enrichment tool in the quantification of human liver endogenous proteins using MRM-based targeted proteomics. These methods expand the current toolbox in target enrichment from tissue samples, and the results will aid monitoring the distribution of future tissue biomarkers and tissue biotherapeutics, and can be used in the development of PBPK models to predict exposure and therapeutic effects of small molecule drugs, as well as biotherapeutics.","abstract_has_math":false,"creators":["Qiu, Xiazi"],"institution":"University of Kansas","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Wang, Michael Z"],"committee_chairs":[],"committee_members":[],"year":2020,"date_issued":"2020-12-31","date_published":"2020-12-31","updated_at":"2026-07-24T02:47:27Z","subjects":["Pharmaceutical sciences","bioanalysis","protein quantification","Subcellular Fractionation","Targeted proteomics","UPLC-MRM"],"languages":["en"],"rights":["Copyright held by the author."],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["http://dissertations.umi.com/ku:17477"],"render_values":[{"text":"http://dissertations.umi.com/ku:17477","href":"http://dissertations.umi.com/ku:17477","code":true}]}]},"links":{"outbound_url":"https://hdl.handle.net/1808/37157","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Wang, Michael Z"]},{"key":"dc:creator","label":"Author","values":["Qiu, Xiazi"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2026-04-13T22:12:20Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2026-04-13T22:12:20Z"]},{"key":"dc:date.issued","label":"Date","values":["2020-12-31"]},{"key":"dc:publisher","label":"Institution","values":["University of Kansas"]},{"key":"dc:type","label":"Dc Type","values":["Dissertation"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Pharmaceutical sciences","bioanalysis","protein quantification","Subcellular Fractionation","Targeted proteomics","UPLC-MRM"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright held by the author."]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["http://dissertations.umi.com/ku:17477"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://hdl.handle.net/1808/37157"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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 workflow as sample enrichment tools to demonstrate their importance in quantifying human liver endogenous proteins. First, human liver neonatal Fc receptor and beta 2-microglobulin were enriched in a post-nuclear supernatant, and a UPLC-MRM method was developed and validated to quantify these two proteins. Our quantitative results and determination of interindividual variability will aid the future development of more accurate PBPK models for immunoglobulin G- or albumin- based biotherapeutics. Second, UPLC-MRM methods were developed for a panel of organelle marker proteins to accelerate the quality assessment of organelles isolated from a sucrose gradient. Lastly, commercially available organelle isolation kits were assessed for their compatibilities and enrichment efficiencies in UPLC-MRM workflows for the targeted quantification of human hepatic drug transporters and xenobiotic nuclear receptors. In summary, this dissertation demonstrates the suitability of subcellular fractionation as a sample enrichment tool in the quantification of human liver endogenous proteins using MRM-based targeted proteomics. These methods expand the current toolbox in target enrichment from tissue samples, and the results will aid monitoring the distribution of future tissue biomarkers and tissue biotherapeutics, and can be used in the development of PBPK models to predict exposure and therapeutic effects of small molecule drugs, as well as biotherapeutics."]},{"key":"dc:title","label":"Title","values":["Subcellular Fractionation Methods for the Quantification of Endogenous Protein in Human Liver Using Targeted Proteomics"]}]}],"canonical_facts":{"dc:contributor.advisor":["Wang, Michael Z"],"dc:creator":["Qiu, Xiazi"],"dc:date.accessioned":["2026-04-13T22:12:20Z"],"dc:date.available":["2026-04-13T22:12:20Z"],"dc:date.issued":["2020-12-31"],"dc:description.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 workflow as sample enrichment tools to demonstrate their importance in quantifying human liver endogenous proteins. First, human liver neonatal Fc receptor and beta 2-microglobulin were enriched in a post-nuclear supernatant, and a UPLC-MRM method was developed and validated to quantify these two proteins. Our quantitative results and determination of interindividual variability will aid the future development of more accurate PBPK models for immunoglobulin G- or albumin- based biotherapeutics. Second, UPLC-MRM methods were developed for a panel of organelle marker proteins to accelerate the quality assessment of organelles isolated from a sucrose gradient. Lastly, commercially available organelle isolation kits were assessed for their compatibilities and enrichment efficiencies in UPLC-MRM workflows for the targeted quantification of human hepatic drug transporters and xenobiotic nuclear receptors. In summary, this dissertation demonstrates the suitability of subcellular fractionation as a sample enrichment tool in the quantification of human liver endogenous proteins using MRM-based targeted proteomics. These methods expand the current toolbox in target enrichment from tissue samples, and the results will aid monitoring the distribution of future tissue biomarkers and tissue biotherapeutics, and can be used in the development of PBPK models to predict exposure and therapeutic effects of small molecule drugs, as well as biotherapeutics."],"dc:identifier.other":["http://dissertations.umi.com/ku:17477"],"dc:identifier.uri":["https://hdl.handle.net/1808/37157"],"dc:language.iso":["en"],"dc:publisher":["University of Kansas"],"dc:rights":["Copyright held by the author."],"dc:subject":["Pharmaceutical sciences","bioanalysis","protein quantification","Subcellular Fractionation","Targeted proteomics","UPLC-MRM"],"dc:title":["Subcellular Fractionation Methods for the Quantification of Endogenous Protein in Human Liver Using Targeted Proteomics"],"dc:type":["Dissertation"]},"updated_at":"2026-07-24T02:47:27Z"}