Multiple-approaches to the identification and quantification of cytochromes P450 in human liver tissue by mass spectrometry.
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 labeled tryptic peptide and analyzed by LC-MS/MS. Using multiple reaction monitoring (MRM) for the isotope-labeled tryptic peptides and their natural unlabeled analogues quantification could be performed over the range of 0.1-1.5 pmol on column. Liver microsomes from four individuals were analyzed for CYP2E1 giving values of 88-200 pmol/mg microsomal protein. The CYP1A2 content of microsomes from a further three individuals ranged from 165 to 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.
Seibert
Cathrin
C
Department of Pharmaceutical and Biological Chemistry, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, United Kingdom.
Davidson
Brian R
BR
Fuller
Barry J
BJ
Patterson
Laurence H
LH
Griffiths
William J
WJ
Wang
Yuqin
Y
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D053719:Tandem Mass Spectrometry / Q000379:methods
D003577:Cytochrome P-450 Enzyme System