Global Proteomic Analysis of Human Liver Microsomes: Rapid Characterization and Quantification of Hepatic Drug-Metabolizing Enzymes

Achour, Brahim; Feteisi, Hajar Al; Lanucara, Francesco; Rostami‐Hodjegan, Amin; Barber, Jill

doi:10.1124/dmd.116.074732

Cited by 42 publications

(69 citation statements)

References 55 publications

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“…The limit of quantification was estimated as previously described (Achour et al . ) at 0.06 fmol of peptide on column (translating to protein abundance of 0.1 fmol/μg protein).…”

Section: Resultsmentioning

confidence: 99%

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Identification and quantification of blood–brain barrier transporters in isolated rat brain microvessels

Feteisi

Al‐Majdoub

Achour

et al. 2018

Journal of Neurochemistry

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The blood-brain barrier (BBB) maintains brain homeostasis by tightly regulating the exchange of molecules with systemic circulation. It consists primarily of microvascular endothelial cells surrounded by astrocytic endfeet, pericytes, and microglia. Understanding the make-up of transporters in rat BBB is essential to the translation of pharmacological and toxicological observations into humans. In this study, experimental workflows are presented in which the optimization of (a) isolation of rat brain microvessels (b) enrichment of endothelial cells, and (c) extraction and digestion of proteins were evaluated, followed by identification and quantification of BBB proteins. Optimization of microvessel isolation was indicated by 15-fold enrichment of endothelial cell marker Glut1 mRNA, whereas markers for other cell types were not enriched. Filter-aided sample preparation was shown to be superior to in-solution sample preparation (10251 peptides vs. 7533 peptides). Label-free proteomics was used to identify nearly 2000 proteins and quantify 1276 proteins in isolated microvessels. A combination of targeted and global proteomics was adopted to measure protein abundance of 6 ATP-binding cassette and 27 solute carrier transporters. Data analysis using proprietary Progenesis and open access MaxQuant software showed overall agreement; however, Abcb9 and Slc22a8 were quantified only by MaxQuant, whereas Abcc9 and Abcd3 were quantified only by Progenesis. Agreement between targeted and untargeted quantification was demonstrated for Abcb1 (19.7 ± 1.4 vs. 17.8 ± 2.3) and Abcc4 (2.2 ± 0.7 vs. 2.1 ± 0.4), respectively. Rigorous quantification of BBB proteins, as reported in this study, should assist with translational modeling efforts involving brain disposition of xenobiotics.

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“…The limit of quantification was estimated as previously described (Achour et al . ) at 0.06 fmol of peptide on column (translating to protein abundance of 0.1 fmol/μg protein).…”

Section: Resultsmentioning

confidence: 99%

“…; Achour et al . ). Details of data analysis and quantification approaches are provided in Appendix S1.…”

Section: Methodsmentioning

confidence: 97%

Identification and quantification of blood–brain barrier transporters in isolated rat brain microvessels

Feteisi

Al‐Majdoub

Achour

et al. 2018

Journal of Neurochemistry

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“…A recent proteomics study has shown that CES1 is the most abundant drug‐metabolizing enzyme in human liver microsomes . As the predominant hepatic hydrolase, CES1 plays a critical role in the activation/deactivation of various medications.…”

Section: Discussionmentioning

confidence: 99%

Functional Study of Carboxylesterase 1 Protein Isoforms

2019

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Carboxylesterase 1 (CES1) is a primary human hepatic hydrolase involved in hydrolytic biotransformation of numerous medications. Considerable interindividual variability in CES1 expression and activity has been consistently reported. Four isoforms of the CES1 protein are produced by alternative splicing (AS). In the current study, the activity and expression of each CES1 isoform are examined using transfected cell lines, and CES1 isoform composition and its impact on CES1 activity in human livers are determined. In transfected cells, isoforms 3 and 4 show mRNA and protein expressions comparable to isoforms 1 and 2, but have significantly impaired activity when hydrolyzing enalapril and clopidogrel. In individual human liver samples, isoforms 1 and 2 are the major forms, contributing 73–90% of the total CES1 protein expression. In addition, the protein expression ratios of isoforms 1 and 2 to isoforms 3 and 4 are positively associated with CES1 activity in the liver, suggesting that CES1 isoform composition is a factor contributing to the variability in hepatic CES1 function. Further investigations of the regulation of CES1 AS would improve the understanding of CES1 variability and help develop a strategy to optimize the pharmacotherapy of many CES1 substrate medications.

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“…20 Peptide separation is achieved on nano-LC, micro-LC, or conventional-LC systems, which can be further improved using ion mobility separation prior to MS analysis. 21,22 Analysis of precursor and product ions (mass filtering) can be achieved in space or in time depending on the mass analyzer. Selection of proteotypic peptides utilizes multiple in silico or experimental approaches and considers recommended criteria to ensure peptides are representative of target proteins ( Table 1).…”

Section: Basic Proteomics Workflowmentioning

confidence: 99%

“…For example, Na + /K + ATPase and calnexin have previously been used as markers for basolateral plasma membrane and reticular microsomal membrane, respectively. 22,31,37 STATE of the ART Membrane enrichment is carried out using various centrifugation or membrane extraction methods, often as commercial kits. A substantial amount of published transporter data are generated using these kits; however, no systematic assessment of the quality of kit-enriched fractions has been reported.…”

Section: Sample Quality and Characteristicsmentioning

confidence: 99%

Toward a Consensus on Applying Quantitative Liquid Chromatography‐Tandem Mass Spectrometry Proteomics in Translational Pharmacology Research: A White Paper

Prasad

Achour

Artursson

et al. 2019

Clin Pharma and Therapeutics

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129

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Quantitative translation of information on drug absorption, disposition, receptor engagement, and drug–drug interactions from bench to bedside requires models informed by physiological parameters that link in vitro studies to in vivo outcomes. To predict in vivo outcomes, biochemical data from experimental systems are routinely scaled using protein quantity in these systems and relevant tissues. Although several laboratories have generated useful quantitative proteomic data using state‐of‐the‐art mass spectrometry, no harmonized guidelines exit for sample analysis and data integration to in vivo translation practices. To address this gap, a workshop was held on September 27 and 28, 2018, in Cambridge, MA, with 100 experts attending from academia, the pharmaceutical industry, and regulators. Various aspects of quantitative proteomics and its applications in translational pharmacology were debated. A summary of discussions and best practices identified by this expert panel are presented in this “White Paper” alongside unresolved issues that were outlined for future debates.

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Global Proteomic Analysis of Human Liver Microsomes: Rapid Characterization and Quantification of Hepatic Drug-Metabolizing Enzymes

Cited by 42 publications

References 55 publications

Identification and quantification of blood–brain barrier transporters in isolated rat brain microvessels

Identification and quantification of blood–brain barrier transporters in isolated rat brain microvessels

Functional Study of Carboxylesterase 1 Protein Isoforms

Toward a Consensus on Applying Quantitative Liquid Chromatography‐Tandem Mass Spectrometry Proteomics in Translational Pharmacology Research: A White Paper

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