Journal article 1065 views
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
,
Yuqin Wang
Journal of Proteome Research, Volume: 8, Issue: 4, Pages: 1672 - 1681
Swansea University Authors:
William Griffiths , Yuqin Wang
Full text not available from this repository: check for access using links below.
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...
| Published in: | Journal of Proteome Research |
|---|---|
| ISSN: | 1535-3893 1535-3907 |
| Published: |
2009
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| Online Access: |
Check full text
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| URI: | https://cronfa.swan.ac.uk/Record/cronfa10946 |
| first_indexed |
2013-07-23T12:04:40Z |
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| last_indexed |
2018-02-09T04:40:13Z |
| id |
cronfa10946 |
| recordtype |
SURis |
| fullrecord |
<?xml version="1.0"?><rfc1807><datestamp>2013-08-01T07:56:37.8887931</datestamp><bib-version>v2</bib-version><id>10946</id><entry>2012-06-05</entry><title>Multiple-Approaches to the Identification and Quantification of Cytochromes P450 in Human Liver Tissue by Mass Spectrometry</title><swanseaauthors><author><sid>3316b1d1b524be1831790933eed1c26e</sid><ORCID>0000-0002-4129-6616</ORCID><firstname>William</firstname><surname>Griffiths</surname><name>William Griffiths</name><active>true</active><ethesisStudent>false</ethesisStudent></author><author><sid>c92729b58622f9fdf6a0e7d8f4ce5081</sid><ORCID>0000-0002-3063-3066</ORCID><firstname>Yuqin</firstname><surname>Wang</surname><name>Yuqin Wang</name><active>true</active><ethesisStudent>false</ethesisStudent></author></swanseaauthors><date>2012-06-05</date><deptcode>MEDS</deptcode><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 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.</abstract><type>Journal Article</type><journal>Journal of Proteome Research</journal><volume>8</volume><journalNumber>4</journalNumber><paginationStart>1672</paginationStart><paginationEnd>1681</paginationEnd><publisher/><placeOfPublication/><issnPrint>1535-3893</issnPrint><issnElectronic>1535-3907</issnElectronic><keywords/><publishedDay>31</publishedDay><publishedMonth>12</publishedMonth><publishedYear>2009</publishedYear><publishedDate>2009-12-31</publishedDate><doi>10.1021/pr800795r</doi><url/><notes/><college>COLLEGE NANME</college><department>Medical School</department><CollegeCode>COLLEGE CODE</CollegeCode><DepartmentCode>MEDS</DepartmentCode><institution>Swansea University</institution><apcterm/><lastEdited>2013-08-01T07:56:37.8887931</lastEdited><Created>2012-06-05T16:28:23.1066672</Created><path><level id="1">Faculty of Medicine, Health and Life Sciences</level><level id="2">Swansea University Medical School - Medicine</level></path><authors><author><firstname>Cathrin</firstname><surname>Seibert</surname><order>1</order></author><author><firstname>Brian R</firstname><surname>Davidson</surname><order>2</order></author><author><firstname>Barry J</firstname><surname>Fuller</surname><order>3</order></author><author><firstname>Laurence H</firstname><surname>Patterson</surname><order>4</order></author><author><firstname>William</firstname><surname>Griffiths</surname><orcid>0000-0002-4129-6616</orcid><order>5</order></author><author><firstname>Yuqin</firstname><surname>Wang</surname><orcid>0000-0002-3063-3066</orcid><order>6</order></author></authors><documents/><OutputDurs/></rfc1807> |
| 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 MEDS 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 Medical School COLLEGE CODE MEDS Swansea University 2013-08-01T07:56:37.8887931 2012-06-05T16:28:23.1066672 Faculty of Medicine, Health and Life Sciences 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 |
Faculty of Medicine, Health and Life Sciences |
| hierarchytype |
|
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facultyofmedicinehealthandlifesciences |
| hierarchy_top_title |
Faculty of Medicine, Health and Life Sciences |
| hierarchy_parent_id |
facultyofmedicinehealthandlifesciences |
| hierarchy_parent_title |
Faculty of Medicine, Health and Life Sciences |
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Swansea University Medical School - Medicine{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Medicine |
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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 |
2009-12-31T06:20:11Z |
| _version_ |
1821385331986923520 |
| score |
11.048171 |