Abstract
Nanoscale reversed-phase liquid chromatography (LC) combined with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) has been used as a method for the direct identification of multiple cytochrome P450 (P450) isoforms found in male and female rat liver. In this targeted proteomic approach, rat liver microsomes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by in-gel tryptic digestion of the proteins present in the 48- to 62-kDa bands. The resultant peptides were extracted and analyzed by LC-ESI-MS/MS. P450 identifications were made by searching the MS/MS data against a rat protein database containing 21,576 entries including 47 P450s using Sequest software (Thermo Electron, Hemel Hempstead, UK). Twenty-four P450 isoforms from the subfamilies 1A, 2A, 2B, 2C, 2D, 2E, 3A, 4A, 4F, CYP17, and CYP19 were positively identified in rat liver.
Footnotes
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↵1 Abbreviations used are: P450, cytochrome P450; MALDI, matrix-assisted laser desorption/ionization; TOF, time-of-flight; PMF, peptide mass fingerprinting; PAGE, polyacrylamide gel electrophoresis; LC, liquid chromatography; ESI, electrospray ionization; MS/MS, tandem mass spectrometry; TFA, trifluoroacetic acid.
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This work is supported by a grant from the Engineering and Physical Sciences Research Council, UK.
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Current address for K.J.W.: University of Hull, Cottingham Road, Hull HU16 7RX, UK.
- Received September 23, 2003.
- Accepted January 13, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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