A Proteomic Approach to the Identification of Cytochrome P450 Isoforms in Male and Female Rat Liver by Nanoscale Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry

Nisar, Sohail; Lane, Catherine; Wilderspin, Andrew F.; Welham, Kevin J.; Griffiths, William J.; Patterson, Laurence H.

doi:10.1124/dmd.32.4.382

Cited by 31 publications

(27 citation statements)

References 36 publications

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“…48 Several efforts to discriminate between CYP isoforms by proteomicsandmassspectrometryaredescribedintheliterature. 15,[47][48][49][50][51][52][53] Most of the studies were performed with rat or mouse liver. But since the CYP pattern of these organisms is mainly different from that in human, studies are hardly comparable.…”

Section: Donormentioning

confidence: 99%

Distinction between Human Cytochrome P450 (CYP) Isoforms and Identification of New Phosphorylation Sites by Mass Spectrometry

et al. 2008

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In mammals, Cytochrome P450 (CYP) enzymes are bound to membranes of the endoplasmic reticulum and mitochondria, where they are responsible for the oxidative metabolism of many xenobiotics as well as organic endogenous compounds. In humans, 57 isoforms were identified which are classified based on sequence homology. In the present work, we demonstrate the performance of a mass spectrometry-based strategy to simultaneously detect and differentiate distinct human Cytochrome P450 (CYP) isoforms including the highly similar CYP3A4, CYP3A5, CYP3A7, as well as CYP2C8, CYP2C9, CYP2C18, CYP2C19, and CYP4F2, CYP4F3, CYP4F11, CYP4F12. Compared to commonly used immunodetection methods, mass spectrometry overcomes limitations such as low antibody specificity and offers high multiplexing possibilities. Furthermore, CYP phosphorylation, which may affect various biochemical and enzymatic properties of these enzymes, is still poorly analyzed, especially in human tissues. Using titanium dioxide resin combined with tandem mass spectrometry for phosphopeptide enrichment and sequencing, we discovered eight human P450 phosphorylation sites, seven of which were novel. The data from surgical human liver samples establish that the isoforms CYP1A2, CYP2A6, CYP2B6, CYP2E1, CYP2C8, CYP2D6, CYP3A4, CYP3A7, and CYP8B1 are phosphorylated in vivo. These results will aid in further investigation of the functional significance of protein phosphorylation for this important group of enzymes.

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Section: Donormentioning

confidence: 99%

Distinction between Human Cytochrome P450 (CYP) Isoforms and Identification of New Phosphorylation Sites by Mass Spectrometry

et al. 2008

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“…Previously we have demonstrated the use of SDS-PAGE in combination with reversed phase LC coupled to electrospray (ES) MS/MS for the identification of multiple P450 proteins (16,17). One of the main advantages of this approach is the molecular mass-based separation provided by SDS-PAGE, which allows proteins only from the molecular mass region of the P450s to be selected for analysis.…”

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confidence: 99%

Comparative Cytochrome P450 Proteomics in the Livers of Immunodeficient Mice Using 18O Stable Isotope Labeling

Lane

Wang

Betts

et al. 2007

Molecular & Cellular Proteomics

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Quantitative changes in cytochrome P450 (CYP) proteins involved in drug metabolism as a consequence of drug treatment are important parameters in predicting the fates and pharmacological consequences of xenobiotics and drugs. In this study we undertook comparative P450 proteomics using liver from control and 1,4-bis-2-(3,5-dichloropyridyloxybenzene) (TCPOBOP)-dosed mice. The method involved separation of microsomal proteins by SDS-PAGE, trypsin digestion, and postdigest 18 O/ 16 O labeling followed by nano-LC-MS/MS for peptide identification and LC-MS for relative quantification. Seventeen P450 proteins were identified from mouse liver of which 16 yielded data sufficient for relative quantification. All the P450s detected were unambiguously identified except the highly homologous CYP2A4/2A5. With the exception of CYP2A12, -2D10, and -2F2, the levels of all the P450s quantified were affected by treatment with TCPOBOP (3 mg/kg). CYP1A2, -2A4/5, -2B10, -2B20, -2C29, -2C37, -2C38, -3A11, and -39A1 were up-regulated, and CYP2C40, -2E1, -3A41, and -27A1 down-regulated. The response of CYP2B20 to stimulation has not been distinguished previously from that of CYP2B10 because of the poor discrimination between these two proteins (they share 87% sequence identity). Differential response to chemical stimulation by closely related members of the CYP2C subfamily was also observed. Molecular & Cellular Proteomics 6:953-962, 2007.The cytochromes P450 (P450s or CYPs 1 ) are a superfamily of mixed function oxidases, members of which are present in virtually all living organisms. P450s are characterized into families and subfamilies by their sequence similarities. Currently there are over 360 families and more than 3100 sequenced and named enzymes; these numbers are continuing to increase. In human there are 57 putatively functional P450 genes, whereas in mouse there are 102 such genes.2 P450s are thought to have evolved, in part, as a protective adaptive response against the toxic effects of environmental chemicals (1). They are the most important drug-metabolizing enzymes in mammals and in humans are responsible for the phase I metabolism of 70 -80% of all clinically used drugs (2). In addition to their detoxification role, P450s can also be responsible for the conversion of chemical toxins and procarcinogens to their toxic or carcinogenic forms (3). The ability of P450s to activate chemical toxins has been exploited in cancer chemotherapy where several anticancer drugs, including cyclophosphamide, ifosfamide, dacarbazine, and AQ4N are known to be metabolically activated by P450 isoforms to their respective cytotoxic species (4). A method that would enable not only the non-selective identification of the P450 enzymes present in diseased tissue but also the quantification of expression levels relative to that of surrounding normal tissue would provide invaluable information regarding the metabolic fate of the selected drugs and hence the outcome of therapy.Traditional approaches to the analysis of P450 enzymes rely on immunoblot...

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“…Rat liver microsomes were obtained from untreated male rats. Previously, it was shown that such microsomes do not contain CYP2B1 and CYP2B2 (Galeva and Altermann, 2002;Galeva et al, 2003;Nisar et al, 2004). Twenty micrograms of total microsomal protein were electrophoresed on 10% SDS-polyacrylamide gel.…”

Section: Resultsmentioning

confidence: 99%

Quantitative Analysis of Cytochrome P450 Isozymes by Means of Unique Isozyme-Specific Tryptic Peptides: A Proteomic Approach

Alterman¹,

Kornilayev²,

Duzhak³

et al. 2005

Drug Metab Dispos

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ABSTRACT:A novel matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry method has been developed to quantitate cytochrome P450 (P450) isozymes based on their unique isozyme-specific tryptic peptides. It was shown that the molar ratio of P450 isozyme-specific peptides is linearly proportional to the mass peak area ratio of corresponding peptides not only in simple two-peptide mixtures, but also in complex digest mixtures. This approach is applicable both to in-gel (as shown for CYP2B1 and CYP2B2) and in-solution digests (as shown for CYP1A2, CYP2E1, and CYP2C19) and does not require introduction of stable isotopes or labeling with isotope-coded affinity tagging. The relative and absolute quantitation can be performed after developing corresponding calibration curves with synthesized P450 isozyme-specific peptide standards. The absolute quantitation of human P450 isozymes was performed by using CYP2B2 isozyme-specific peptide (1306.7 Da) as the universal internal standard. The utility of this approach was demonstrated for two highly homologous (>97%) rat liver CYP2B1 and CYP2B2 and three human P450 isozymes belonging to two different families and three different subfamilies: CYP1A2, CYP2E1, and CYP2C19. In summary, we have demonstrated that MALDI TOF-based peptide mass fingerprinting of different cytochrome P450 isozymes can provide not only qualitative but quantitative data, too.The superfamily of cytochrome(s) P450 (P450) plays a key role in hepatic and extrahepatic drug metabolism, and qualitative and quantitative analyses of the P450 isozyme expression in a particular organ are critical in predicting a metabolic fate of a particular drug or in examination of the potential drug-drug interaction. The number of sequenced and named different P450 isozymes surpassed 3100 (dnelson.utmem.edu/CytochromeP450.html), and the degree of sequence homology, particularly among P450s belonging to the same subfamily, is high (Nelson et al., 1996). None of the existing research approaches to the analysis of individual P450 forms, which include specific P450 inhibitors (Halpert et al., 1994;Kobayashi et al., 2003) or substrates (Kobayashi et al., 2002;Stresser et al., 2002), antibodybased identification (Gelboin et al., 1999;Shou et al., 2000), and mRNA-based analysis (Chow et al., 1999;Zhang et al., 1999), is in a position to provide reliable quantitative and qualitative information on the individual P450 composition in a given type of microsomes. First, only a minority of known P450 isozymes is fully characterized by substrate specificity, and since they exhibit a broad, often overlapping substrate specificity, there is no known substrate or inhibitor that is absolutely specific for an individual P450 isozyme. Second, the high degree of sequence homology among members of the P450 superfamily confounds high specificity of antibody-based analysis, particularly among members of the same subfamily. Third, the application of a quantitative mRNA analysis for the evaluation of P450 isozyme expression...

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A Proteomic Approach to the Identification of Cytochrome P450 Isoforms in Male and Female Rat Liver by Nanoscale Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry

Cited by 31 publications

References 36 publications

Distinction between Human Cytochrome P450 (CYP) Isoforms and Identification of New Phosphorylation Sites by Mass Spectrometry

Distinction between Human Cytochrome P450 (CYP) Isoforms and Identification of New Phosphorylation Sites by Mass Spectrometry

Comparative Cytochrome P450 Proteomics in the Livers of Immunodeficient Mice Using 18O Stable Isotope Labeling

Quantitative Analysis of Cytochrome P450 Isozymes by Means of Unique Isozyme-Specific Tryptic Peptides: A Proteomic Approach

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