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Multiple-Approaches to the Identification and Quantification of Cytochromes P450 in Human Liver Tissue by Mass Spectrometry / Cathrin Seibert, Brian R Davidson, Barry J Fuller, Laurence H Patterson, William Griffiths, Yuqin Wang

Journal of Proteome Research, Volume: 8, Issue: 4, Pages: 1672 - 1681

Swansea University Authors: William Griffiths, Yuqin Wang

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DOI (Published version): 10.1021/pr800795r

Abstract

Here we report the identification and approximate quantification of cytochrome P450 (CYP) proteins in human liver microsomes as determined by nano-LC-MS/MS with application of the exponentially modified protein abundance index (emPAI) algorithm during database searching. Protocols based on 1D-gel pr...

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Published in: Journal of Proteome Research
ISSN: 1535-3893 1535-3907
Published: 2009
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URI: https://cronfa.swan.ac.uk/Record/cronfa10946
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spelling 2013-08-01T07:56:37.8887931 v2 10946 2012-06-05 Multiple-Approaches to the Identification and Quantification of Cytochromes P450 in Human Liver Tissue by Mass Spectrometry 3316b1d1b524be1831790933eed1c26e 0000-0002-4129-6616 William Griffiths William Griffiths true false c92729b58622f9fdf6a0e7d8f4ce5081 0000-0002-3063-3066 Yuqin Wang Yuqin Wang true false 2012-06-05 BMS Here we report the identification and approximate quantification of cytochrome P450 (CYP) proteins in human liver microsomes as determined by nano-LC-MS/MS with application of the exponentially modified protein abundance index (emPAI) algorithm during database searching. Protocols based on 1D-gel protein separation and 2D-LC peptide separation gave comparable results. In total 18 CYP isoforms were unambiguously identified based on unique peptide matches. Further, we have determined the absolute quantity of two CYP enzymes (2E1 and 1A2) in human liver microsomes using stable-isotope dilution mass spectrometry, where microsomal proteins were separated by 1D-gel electrophoresis, digested with trypsin in the presence of either a CYP2E1- or 1A2-specific stable-isotope labelled tryptic peptide and analysed by LC-MS/MS. Using multiple reaction monitoring (MRM) for the isotope-labelled tryptic peptides and their natural unlabelled analogues quantification could be performed over the range of 0.1 – 1.5 pmol on column. Liver microsomes from four individuals were analysed for CYP2E1 giving values of 88 - 200 pmol/mg microsomal protein. The CYP1A2 content of microsomes from a further three individuals ranged from 165 – 263 pmol/mg microsomal protein. Although, in this proof-of-concept study for CYP quantification, the two CYP-isoforms were quantified from different samples, there are no practical reasons to prevent multiplexing the method to allow the quantification of multiple CYP-isoforms in a single sample. Journal Article Journal of Proteome Research 8 4 1672 1681 1535-3893 1535-3907 31 12 2009 2009-12-31 10.1021/pr800795r COLLEGE NANME Biomedical Sciences COLLEGE CODE BMS Swansea University 2013-08-01T07:56:37.8887931 2012-06-05T16:28:23.1066672 Swansea University Medical School Medicine Cathrin Seibert 1 Brian R Davidson 2 Barry J Fuller 3 Laurence H Patterson 4 William Griffiths 0000-0002-4129-6616 5 Yuqin Wang 0000-0002-3063-3066 6
title Multiple-Approaches to the Identification and Quantification of Cytochromes P450 in Human Liver Tissue by Mass Spectrometry
spellingShingle Multiple-Approaches to the Identification and Quantification of Cytochromes P450 in Human Liver Tissue by Mass Spectrometry
William, Griffiths
Yuqin, Wang
title_short Multiple-Approaches to the Identification and Quantification of Cytochromes P450 in Human Liver Tissue by Mass Spectrometry
title_full Multiple-Approaches to the Identification and Quantification of Cytochromes P450 in Human Liver Tissue by Mass Spectrometry
title_fullStr Multiple-Approaches to the Identification and Quantification of Cytochromes P450 in Human Liver Tissue by Mass Spectrometry
title_full_unstemmed Multiple-Approaches to the Identification and Quantification of Cytochromes P450 in Human Liver Tissue by Mass Spectrometry
title_sort Multiple-Approaches to the Identification and Quantification of Cytochromes P450 in Human Liver Tissue by Mass Spectrometry
author_id_str_mv 3316b1d1b524be1831790933eed1c26e
c92729b58622f9fdf6a0e7d8f4ce5081
author_id_fullname_str_mv 3316b1d1b524be1831790933eed1c26e_***_William, Griffiths
c92729b58622f9fdf6a0e7d8f4ce5081_***_Yuqin, Wang
author William, Griffiths
Yuqin, Wang
author2 Cathrin Seibert
Brian R Davidson
Barry J Fuller
Laurence H Patterson
William Griffiths
Yuqin Wang
format Journal article
container_title Journal of Proteome Research
container_volume 8
container_issue 4
container_start_page 1672
publishDate 2009
institution Swansea University
issn 1535-3893
1535-3907
doi_str_mv 10.1021/pr800795r
college_str Swansea University Medical School
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hierarchy_top_id swanseauniversitymedicalschool
hierarchy_top_title Swansea University Medical School
hierarchy_parent_id swanseauniversitymedicalschool
hierarchy_parent_title Swansea University Medical School
department_str Medicine{{{_:::_}}}Swansea University Medical School{{{_:::_}}}Medicine
document_store_str 0
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description Here we report the identification and approximate quantification of cytochrome P450 (CYP) proteins in human liver microsomes as determined by nano-LC-MS/MS with application of the exponentially modified protein abundance index (emPAI) algorithm during database searching. Protocols based on 1D-gel protein separation and 2D-LC peptide separation gave comparable results. In total 18 CYP isoforms were unambiguously identified based on unique peptide matches. Further, we have determined the absolute quantity of two CYP enzymes (2E1 and 1A2) in human liver microsomes using stable-isotope dilution mass spectrometry, where microsomal proteins were separated by 1D-gel electrophoresis, digested with trypsin in the presence of either a CYP2E1- or 1A2-specific stable-isotope labelled tryptic peptide and analysed by LC-MS/MS. Using multiple reaction monitoring (MRM) for the isotope-labelled tryptic peptides and their natural unlabelled analogues quantification could be performed over the range of 0.1 – 1.5 pmol on column. Liver microsomes from four individuals were analysed for CYP2E1 giving values of 88 - 200 pmol/mg microsomal protein. The CYP1A2 content of microsomes from a further three individuals ranged from 165 – 263 pmol/mg microsomal protein. Although, in this proof-of-concept study for CYP quantification, the two CYP-isoforms were quantified from different samples, there are no practical reasons to prevent multiplexing the method to allow the quantification of multiple CYP-isoforms in a single sample.
published_date 2009-12-31T03:22:42Z
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