Journal article 946 views
Multiple-Approaches to the Identification and Quantification of Cytochromes P450 in Human Liver Tissue by Mass Spectrometry
,
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 |
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| ISSN: | 1535-3893 1535-3907 |
| Published: |
2009
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| Online Access: |
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| URI: | https://cronfa.swan.ac.uk/Record/cronfa10946 |
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| 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. |
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| College: |
Faculty of Medicine, Health and Life Sciences |
| Issue: |
4 |
| Start Page: |
1672 |
| End Page: |
1681 |