Precolumn derivatization of cysteine residues for quantitative analysis of five major cytochrome P450 isoenzymes by liquid chromatography/tandem mass spectrometry.
The development of a novel method for absolute quantification of the five most clinically relevant CYP450 isoenzymes is described based on chemical derivatization of cysteine residues. The sulfhydryl-reactive reagents, 2-bromo-4'-chloroacetophenone (p-CPB) and 2-bromo-4'-bromoacetophenone (p-BPB), are proposed for use in quantitative proteomics. After reducing and denaturing, the P450s are derivatized with p-CPB for sulfhydryl alkylation then subjected to trypsin digestion. The resulting p-CPB-attached peptides are enriched using a phenyl resin solid-phase cartridge, then separated on a Zorbax 300SB reversed-phase column, and detected under positive electrospray ionization in the multiple reaction monitoring mode. Quantification is achieved using p-BPB-modified peptides as internal standards. Validation results demonstrated that this method showed good linearity between the concentration range of 10 fmol/microg to 5 pmol/microg for the six selected peptides in a complex matrix (rat liver microsomal protein). Intra-day and inter-day precision, expressed by relative standard deviation, were all less than 18%. Assay accuracy was within +/- 20% in terms of relative error. The quantitative derivatization approach proved to be reproducible, cost-effective and readily suitable for high-throughput assays. The reliability of this method for quantification of intact P450s was demonstrated through comparing with the well-applied isotope-coded affinity tag (ICAT) method.
Duan
Xiaotao
X
Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang, Liaoning 110016, China.
Chen
Xiaoyan
X
Yang
Yiming
Y
Zhong
Dafang
D
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D007527:Isoenzymes / Q000737:chemistry; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods*; D051381:Rats; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*