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Subcellular fractionation of human liver reveals limits in global proteomic quantification from isolated fractions
Max Planck Inst Biochem, Dept Prote & Signal Transduct, Biochem Prote Grp, D-82152 Martinsried, Germany..
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. AstraZeneca, Innovat Med & Early Dev Biotech Unit, Cardiovasc & Metab Dis, Pepparedsleden 1, S-43183 Molndal, Sweden..ORCID iD: 0000-0002-2810-7518
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
2016 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 509, p. 82-88Article in journal (Refereed) Published
Abstract [en]

The liver plays an important role in metabolism and elimination of xenobiotics, including drugs. Determination of concentrations of proteins involved in uptake, distribution, metabolism, and excretion of xenobiotics is required to understand and predict elimination mechanisms in this tissue. In this work, we have fractionated homogenates of snap -frozen human liver by differential centrifugation and performed quantitative mass spectrometry -based proteomic analysis of each fraction. Concentrations of proteins were calculated by the "total protein approach". A total of 4586 proteins were identified by at least five peptides and were quantified in all fractions. We found that the xenobiotics transporters of the canalicular and basolateral membranes were differentially enriched in the subcellular fractions and that phase I and II metabolizing enzymes, the cytochrome P450s and the UDP glucuronyl transferases, have complex subcellular distributions. These findings show that there is no simple way to scale the data from measurements in arbitrarily selected membrane fractions using a single scaling factor for all the proteins of interest. This study also provides the first absolute quantitative subcellular catalog of human liver proteins obtained from frozen tissue specimens. Our data provide quantitative insights into the sub cellular distribution of proteins and can be used as a guide for development of fractionation procedures.

Place, publisher, year, edition, pages
2016. Vol. 509, p. 82-88
Keywords [en]
Human liver, Subcellular fractionation, Absolute quantitative proteomics, Total protein approach, Drug metabolism, Drug transport
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:uu:diva-303255DOI: 10.1016/j.ab.2016.06.006ISI: 000380866800013PubMedID: 27311553OAI: oai:DiVA.org:uu-303255DiVA, id: diva2:971502
Funder
Swedish Research Council, 2822Available from: 2016-09-16 Created: 2016-09-15 Last updated: 2019-07-26Bibliographically approved
In thesis
1. Proteomics-informed analysis of drug disposition in the human liver and small intestine
Open this publication in new window or tab >>Proteomics-informed analysis of drug disposition in the human liver and small intestine
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Orally administered drugs are absorbed in the intestine and generally metabolized in the liver. Therefore, understanding factors determining drug distribution and elimination in these tissues is important. This thesis aimed at using mass spectrometry (MS)-based proteomics and functional studies to better understand in vitro model systems used for drug clearance predictions. Further, it aimed at understanding the changes in drug disposition caused by obesity and gastric bypass surgery (GBP).

The study was initiated by investigating factors influencing MS-based protein quantification by comparing results from different proteomics methods, and by studying protein distribution during subcellular fractionation. The largest variability in protein quantification was ascribed to insufficient enrichment from subcellular fractionation, most likely due to collection of the majority of the proteins in the initial fraction of the fractionation protocols.

Proteomics and metabolic activity analyses were then used to investigate differences in intrinsic clearance from two commonly used in vitro systems, human liver microsomes and hepatocytes. For some compounds, the faster microsomal metabolism could be explained by a higher available unbound drug concentration and CYP content in the microsomes as compared to in the hepatocytes.

Next, inter-individual protein expression variability in human liver and jejunum was explored. This showed that proteins covered a wide inter-individual variability spectrum, in which proteins with low variabilities were associated with essential cellular functions, while many proteins with high variabilities were disease-related.

Further, the effects of obesity, GBP, and weight loss on the proteomes of human liver and jejunum were analyzed. After GBP and subsequent weight loss, patients showed lower levels of jejunal proteins involved in inflammatory response and drug metabolism.

Finally, proteomics data from patients with and without obesity was combined with parameters from in vitro transport kinetics, and a mechanistic model to predict drug disposition was developed. The model successfully predicted rosuvastatin plasma concentrations in the patients.

In conclusion, this thesis has provided insights into factors influencing protein quantification and function in vitro. Furthermore, this thesis demonstrates how proteomics contributes to improved understanding of inter-individual and physiological differences, and how it can be used for in vitro-in vivo scaling of drug clearance.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 79
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 273
Keywords
proteomics, protein concentration, drug disposition, drug transport, drug metabolism, human small intestine, human liver, human hepatocytes, human liver microsomes, inter-individual variability, drug clearance, obesity, prediction model
National Category
Pharmaceutical Sciences
Research subject
Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-389741 (URN)978-91-513-0694-0 (ISBN)
Public defence
2019-09-13, B42, Biomedical center, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2019-08-22 Created: 2019-07-26 Last updated: 2019-08-22

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