Optimization and validation of a label-free MRM method for the quantification of cytochrome P450 isoforms in biological samples
Cytochromes P450 (CYPs) play critical roles in oxidative metabolism of many endogenous and exogenous compounds. Protein expression levels of CYPs in liver provide relevant information for a better understanding of the importance of CYPs in pharmacology and toxicology. This work aimed at establishing...
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| Veröffentlicht in: | Analytical and bioanalytical chemistry 2014-08, Vol.406 (20), p.4861-4874 |
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| creator | Al Ali, Ahmad Touboul, David Le Caër, Jean-Pierre Schmitz-Afonso, Isabelle Flinois, Jean-Pierre Marchetti, Catherine De Waziers, Isabelle Brunelle, Alain Laprévote, Olivier Beaune, Philippe |
| description | Cytochromes P450 (CYPs) play critical roles in oxidative metabolism of many endogenous and exogenous compounds. Protein expression levels of CYPs in liver provide relevant information for a better understanding of the importance of CYPs in pharmacology and toxicology. This work aimed at establishing a simple method to quantify six CYPs (CYP3A4, CYP3A5, CYP1A2, CYP2D6, CYP2C9, and CYP2J2) in various biological samples without isotopic labeling. The biological matrix was spiked with the standard peptides prior to the digestion step to realize a label-free quantification by mass spectrometry. The method was validated and applied to quantify these six isoforms in both human liver microsomes and mitochondria, but also in recombinant expression systems such as baculosomes and the HepG2 cell line. The results showed intra-assay and interassay accuracy and precision within 16 % and 5 %, respectively, at the low quality control level, and demonstrated the advantages of the method in terms of reproducibility and cost.
Figure
Calibration curve in complex matrix for CYPs quantification |
| doi_str_mv | 10.1007/s00216-014-7928-z |
| format | Article |
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Figure
Calibration curve in complex matrix for CYPs quantification</description><identifier>ISSN: 1618-2642</identifier><identifier>EISSN: 1618-2650</identifier><identifier>DOI: 10.1007/s00216-014-7928-z</identifier><identifier>PMID: 24952904</identifier><language>eng</language><publisher>Berlin/Heidelberg: Springer Berlin Heidelberg</publisher><subject>Analysis ; Analytical Chemistry ; Biochemistry ; Biological ; Biological samples ; Brain ; Characterization and Evaluation of Materials ; Chemical Sciences ; Chemistry ; Chemistry and Materials Science ; Cysteine - chemistry ; Cytochrome ; Cytochrome P-450 ; Cytochrome P-450 Enzyme System - metabolism ; Cytochromes P450 ; Digestion ; Enzymes ; Fluorescence ; Fluorescence microscopy ; Food Science ; Hep G2 Cells ; Humans ; Immunoassays ; Isoenzymes ; Labeling ; Laboratory Medicine ; Liver ; Mass spectrometry ; Measurement ; Metabolism ; Methods ; Microsomes, Liver - enzymology ; Mitochondria ; Mitochondria, Liver - enzymology ; Monitoring/Environmental Analysis ; Optimization ; Organic chemistry ; Peptide Fragments - analysis ; Peptides ; Pharmacology ; Physiological aspects ; Protein expression ; Proteins ; Quality control ; Recombinant ; Recombinant Proteins - metabolism ; Reproducibility ; Research Paper ; Scientific imaging ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods ; Toxicology</subject><ispartof>Analytical and bioanalytical chemistry, 2014-08, Vol.406 (20), p.4861-4874</ispartof><rights>Springer-Verlag Berlin Heidelberg 2014</rights><rights>COPYRIGHT 2014 Springer</rights><rights>Distributed under a Creative Commons Attribution 4.0 International License</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c618t-7bcebf4640db9858c64e58e7f585344fcc51ddd81ec69558d585b2672f301ee43</citedby><cites>FETCH-LOGICAL-c618t-7bcebf4640db9858c64e58e7f585344fcc51ddd81ec69558d585b2672f301ee43</cites><orcidid>0000-0003-2751-774X ; 0000-0003-0665-0074 ; 0000-0001-6526-6481</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s00216-014-7928-z$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00216-014-7928-z$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>230,315,781,785,886,27929,27930,41493,42562,51324</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24952904$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://hal.science/hal-01024697$$DView record in HAL$$Hfree_for_read</backlink></links><search><creatorcontrib>Al Ali, Ahmad</creatorcontrib><creatorcontrib>Touboul, David</creatorcontrib><creatorcontrib>Le Caër, Jean-Pierre</creatorcontrib><creatorcontrib>Schmitz-Afonso, Isabelle</creatorcontrib><creatorcontrib>Flinois, Jean-Pierre</creatorcontrib><creatorcontrib>Marchetti, Catherine</creatorcontrib><creatorcontrib>De Waziers, Isabelle</creatorcontrib><creatorcontrib>Brunelle, Alain</creatorcontrib><creatorcontrib>Laprévote, Olivier</creatorcontrib><creatorcontrib>Beaune, Philippe</creatorcontrib><title>Optimization and validation of a label-free MRM method for the quantification of cytochrome P450 isoforms in biological samples</title><title>Analytical and bioanalytical chemistry</title><addtitle>Anal Bioanal Chem</addtitle><addtitle>Anal Bioanal Chem</addtitle><description>Cytochromes P450 (CYPs) play critical roles in oxidative metabolism of many endogenous and exogenous compounds. Protein expression levels of CYPs in liver provide relevant information for a better understanding of the importance of CYPs in pharmacology and toxicology. This work aimed at establishing a simple method to quantify six CYPs (CYP3A4, CYP3A5, CYP1A2, CYP2D6, CYP2C9, and CYP2J2) in various biological samples without isotopic labeling. The biological matrix was spiked with the standard peptides prior to the digestion step to realize a label-free quantification by mass spectrometry. The method was validated and applied to quantify these six isoforms in both human liver microsomes and mitochondria, but also in recombinant expression systems such as baculosomes and the HepG2 cell line. The results showed intra-assay and interassay accuracy and precision within 16 % and 5 %, respectively, at the low quality control level, and demonstrated the advantages of the method in terms of reproducibility and cost.
Figure
Calibration curve in complex matrix for CYPs quantification</description><subject>Analysis</subject><subject>Analytical Chemistry</subject><subject>Biochemistry</subject><subject>Biological</subject><subject>Biological samples</subject><subject>Brain</subject><subject>Characterization and Evaluation of Materials</subject><subject>Chemical Sciences</subject><subject>Chemistry</subject><subject>Chemistry and Materials Science</subject><subject>Cysteine - chemistry</subject><subject>Cytochrome</subject><subject>Cytochrome P-450</subject><subject>Cytochrome P-450 Enzyme System - metabolism</subject><subject>Cytochromes P450</subject><subject>Digestion</subject><subject>Enzymes</subject><subject>Fluorescence</subject><subject>Fluorescence microscopy</subject><subject>Food Science</subject><subject>Hep G2 Cells</subject><subject>Humans</subject><subject>Immunoassays</subject><subject>Isoenzymes</subject><subject>Labeling</subject><subject>Laboratory Medicine</subject><subject>Liver</subject><subject>Mass spectrometry</subject><subject>Measurement</subject><subject>Metabolism</subject><subject>Methods</subject><subject>Microsomes, Liver - enzymology</subject><subject>Mitochondria</subject><subject>Mitochondria, Liver - enzymology</subject><subject>Monitoring/Environmental Analysis</subject><subject>Optimization</subject><subject>Organic chemistry</subject><subject>Peptide Fragments - analysis</subject><subject>Peptides</subject><subject>Pharmacology</subject><subject>Physiological aspects</subject><subject>Protein expression</subject><subject>Proteins</subject><subject>Quality control</subject><subject>Recombinant</subject><subject>Recombinant Proteins - metabolism</subject><subject>Reproducibility</subject><subject>Research Paper</subject><subject>Scientific imaging</subject><subject>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - 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and validation of a label-free MRM method for the quantification of cytochrome P450 isoforms in biological samples</title><author>Al Ali, Ahmad ; Touboul, David ; Le Caër, Jean-Pierre ; Schmitz-Afonso, Isabelle ; Flinois, Jean-Pierre ; Marchetti, Catherine ; De Waziers, Isabelle ; Brunelle, Alain ; Laprévote, Olivier ; Beaune, Philippe</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c618t-7bcebf4640db9858c64e58e7f585344fcc51ddd81ec69558d585b2672f301ee43</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Analysis</topic><topic>Analytical Chemistry</topic><topic>Biochemistry</topic><topic>Biological</topic><topic>Biological samples</topic><topic>Brain</topic><topic>Characterization and Evaluation of Materials</topic><topic>Chemical Sciences</topic><topic>Chemistry</topic><topic>Chemistry and Materials Science</topic><topic>Cysteine - chemistry</topic><topic>Cytochrome</topic><topic>Cytochrome P-450</topic><topic>Cytochrome P-450 Enzyme System - metabolism</topic><topic>Cytochromes P450</topic><topic>Digestion</topic><topic>Enzymes</topic><topic>Fluorescence</topic><topic>Fluorescence microscopy</topic><topic>Food Science</topic><topic>Hep G2 Cells</topic><topic>Humans</topic><topic>Immunoassays</topic><topic>Isoenzymes</topic><topic>Labeling</topic><topic>Laboratory Medicine</topic><topic>Liver</topic><topic>Mass spectrometry</topic><topic>Measurement</topic><topic>Metabolism</topic><topic>Methods</topic><topic>Microsomes, Liver - enzymology</topic><topic>Mitochondria</topic><topic>Mitochondria, Liver - enzymology</topic><topic>Monitoring/Environmental Analysis</topic><topic>Optimization</topic><topic>Organic chemistry</topic><topic>Peptide Fragments - analysis</topic><topic>Peptides</topic><topic>Pharmacology</topic><topic>Physiological aspects</topic><topic>Protein 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oxidative metabolism of many endogenous and exogenous compounds. Protein expression levels of CYPs in liver provide relevant information for a better understanding of the importance of CYPs in pharmacology and toxicology. This work aimed at establishing a simple method to quantify six CYPs (CYP3A4, CYP3A5, CYP1A2, CYP2D6, CYP2C9, and CYP2J2) in various biological samples without isotopic labeling. The biological matrix was spiked with the standard peptides prior to the digestion step to realize a label-free quantification by mass spectrometry. The method was validated and applied to quantify these six isoforms in both human liver microsomes and mitochondria, but also in recombinant expression systems such as baculosomes and the HepG2 cell line. The results showed intra-assay and interassay accuracy and precision within 16 % and 5 %, respectively, at the low quality control level, and demonstrated the advantages of the method in terms of reproducibility and cost.
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Calibration curve in complex matrix for CYPs quantification</abstract><cop>Berlin/Heidelberg</cop><pub>Springer Berlin Heidelberg</pub><pmid>24952904</pmid><doi>10.1007/s00216-014-7928-z</doi><tpages>14</tpages><orcidid>https://orcid.org/0000-0003-2751-774X</orcidid><orcidid>https://orcid.org/0000-0003-0665-0074</orcidid><orcidid>https://orcid.org/0000-0001-6526-6481</orcidid></addata></record> |
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| subjects | Analysis Analytical Chemistry Biochemistry Biological Biological samples Brain Characterization and Evaluation of Materials Chemical Sciences Chemistry Chemistry and Materials Science Cysteine - chemistry Cytochrome Cytochrome P-450 Cytochrome P-450 Enzyme System - metabolism Cytochromes P450 Digestion Enzymes Fluorescence Fluorescence microscopy Food Science Hep G2 Cells Humans Immunoassays Isoenzymes Labeling Laboratory Medicine Liver Mass spectrometry Measurement Metabolism Methods Microsomes, Liver - enzymology Mitochondria Mitochondria, Liver - enzymology Monitoring/Environmental Analysis Optimization Organic chemistry Peptide Fragments - analysis Peptides Pharmacology Physiological aspects Protein expression Proteins Quality control Recombinant Recombinant Proteins - metabolism Reproducibility Research Paper Scientific imaging Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods Toxicology |
| title | Optimization and validation of a label-free MRM method for the quantification of cytochrome P450 isoforms in biological samples |
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