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Modern methods of cytochrome P450 analysis

Article in Biochemistry (Moscow) Supplement Series B Biomedical Chemistry · April 2013

DOI: 10.1134/S1990750813020078

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ISSN 19907508, Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry, 2013, Vol. 7, No. 2, pp. 124–135. © Pleiades Publishing, Ltd., 2013.
Original Russian Text © N.E. Moskaleva, V.G. Zgoda, 2013, published in Biomeditsinskaya Khimiya.

Modern Methods of Cytochrome P450 Analysis

N. E. Moskaleva1 and V. G. Zgoda
Orekhovich Institute of Biomedical Chemistry of Russian Academy of Medical Sciences,
ul. Pogodinskaya 10, Moscow, 119121 Russia
email: nemoskaleva@gmail.com
Received October 1, 2012

Abstract—The review describes recent approaches used for evaluation of cytochrome P450 concentration
and activity. It considers employment of modern methods of proteomic analysis including electrophoresis
and chromatography/massspectrometry (MS) for investigation of the particular group of proteins. Special
attention is paid to targeted quantitative MS analysis of cytochromes P450 in biological samples. Finally, we
analyze methods used for assay of cytochromes P450 activities and problems of correlation between content
of certain P450 isoforms and their specific enzymatic activities.

Keywords: quantitative proteomics, massspectrometry, cytochrome P450.

DOI: 10.1134/S1990750813020078

1
INTRODUCTION CYP2D6, CYP2E1, catalyze about 90% of hydroxyla
tion of drug substances [4].
Problems of optimization and personification of
pharmacotherapy become especially important now. Activity of the monooxygenase system towards par
There is increasing evidence that differences in the ticular drug is mainly determined by concentration
rate of drug metabolism observed in various people and functional capacity (i.e. activity of cytochrome
often represent a cause of inadequate pharmacological P450 isoforms specific for this drug). Other compo
responses to administration of drugs and so develop nents of the monooxygenase system, NADPHdepen
ment of personified medicine is impossible without dent reductase and cytochrome b5 usually do not rep
studies of drug metabolism and responses of the body resent ratelimiting factors in monooxygenase reac
to the administered drug [1].
tions [2]. Consequently, individual features of drug
Enzymes of the cytochrome P450 superfamily play metabolism are determined by the personal profile,
a key role in metabolism of xenobiotics [2]. They are concentration and activity of cytochromes P450 [1].
hemecontaining monooxygenases, components
of the microsomal monooxygenase system. Besides Chemical compounds, particularly drug sub
cytochrome P450 (CYP) this system localized stances, may influence content and activity of cyto
onto membranes of the endoplasmic reticulum chromes P450 by increasing their functional capacity
includes NADPH cytochrome P450 reductase and (induction) or decreasing it (inhibition). Features of
cytochrome b5 [2]. enzyme inducers and inhibitors may be attributed not
only to drug substances but also to food components,
Cytochromes P450 are widely distributed in nature
and found in all aerobic organisms. Genomic air and water contaminants, compounds present in
sequences have been obtained for more than tobacco smoke, alcohol [1, 4]. In addition, studies of
12400 cytochromes P450, which represent tens and drug⎯drug interaction at the level of altered activity of
hundreds of families and subfamilies, respectively [3]. the monooxygenase system are also important for
Cytochrome P450 isoenzymes, members of various development of novel drugs.
families and subfamilies, differ by substrate specificity Development of pharmacotherapy and its personi
and regulation of their activity (by inhibitors and
fication require creation of models for studies of drug
inducers); however, some of them may share substrate
specificity, inhibitor and inducer sensitivity. Fifty eight effects and responses of the body. These models need
isoforms of cytochrome P450 have been identified in selective isoformspecific quantitative analysis of
humans and 12 of them are involved in xenobiotic cytochrome P450 in biological objects for determina
metabolism [4]. Isoenzymes of families I, II, and III, tion of ranges in variations of enzyme concentrations
particularly CYP3A4, CYP1A2, CYP2C9, CYP2C19, under normal conditions and during exposure to vari
ous factors and also elucidation of links between con
1 To whom correspondence should be addressed.
tent of cytochromes P450 and their activity.

124
 MODERN METHODS OF CYTOCHROME P450 ANALYSIS 125

In this review we have considered methods used for belonging to the same subfamily, such as CYP3A4 and
determination of proteins of the cytochrome P450 CYP3A7 [12].
superfamily and their activity in biological objects. Alterman et al. [13] developed the method of quan
titative analysis of human CYP2E1, CYP1A2, and
CYP2C19 by means of immunochemical approaches
1. METHODS OF CYTOCHROME P450 and mass spectrometry. This method was tested using
ANALYSIS IN BIOLOGICAL OBJECTS a mixture of recombinant proteins. For each protein
authors selected peptides with unique sequences used
1.1. Differential Spectroscopy for both antibody generation and quantitative analysis
by MALDITOF/MS. Authors indicated difficulties
Differential spectroscopy was one of the first meth of determination of protein concentration by
ods used for determination of cytochrome P450 con MALDITOF/MS associated with heterogeneity of
tent; it is based on analysis of the characteristic spec sample crystallization, ionization quenching by matrix
troscopic shift that appears due to CO binding to components and possibility of spectrum overlap in the
reduced heme iron [5, 6]. Using differential spectros presence of peptides with similar masses in the sample.
copy it is possible to determine content of catalytically Although some antibodies recognizing targeted
active forms of cytochrome P450 in complex biologi CYP isoforms are commercially available authors
cal systems without enzyme isolation. Although this [12⎯14] concluded that direct and simultaneous
method is stable and widely used it has serious limita quantitative determination of cytochromes P450 of
tions. First, accurate measurements require high con the same subfamily still represent a difficult task. For
centrations of cytochrome P450. In addition, using example, generation of isoformspecific antibodies to
differential spectroscopy it is possible to determine cytochromes P450 is complicated by high homology
total concentration of cytochromes P450 but it is inap observed within both one family of cytochromes and
plicable for differential isoformspecific analysis [5]. also between families as well. In addition, availability
of highly purified protein for calibration curves is an
ultimate precondition for determination of CYP con
1.2. Evaluation of Cytochrome P450 Expression centrations by immunological methods [14].
by Corresponding mRNA Levels
Although protein expression was evaluated in
[7⎯10] by analysis of corresponding mRNAs authors 2. METHODS OF PROTEOMIC ANALYSIS
indicated frequent poor correlation between levels of OF CYTOCHROME P450
mRNA and corresponding protein as protein content Usually, proteomic methods of analysis include
is regulated by posttranscriptional and translational stages of preliminary separation of proteins or peptides
mechanisms [7⎯10]. For example, comparison of lung and subsequent identification by mass spectrometry or
cytochrome P450 expression by mRNA, catalytic by immunological approaches [15].
activity and analysis of particular protein level revealed
contradictory results for CYP1A2, CYP2C, and
CYP2D6 [8]. Moreover, in some cases presence and 2.1. Methods Used for Preliminary Separation
content of mRNA does not necessarily imply the pres of Cytochrome P450 Superfamily Proteins in Proteomics
ence of corresponding protein [11].
Electrophoretic methods are widely used for direct
analysis of proteins in biological systems and also for
1.3 Immunological Method of Analysis preliminary fractionation of samples prior MS deter
mination. Authors in [16–21] used twodimensional
Immunological methods such as ELISA or Western (2DE) and onedimensional (1DE) electrophoresis
blotting are more informative methods of quantitative for separation of hydrophobic membrane proteins
analysis of cytochromes P450. According to studies by including cytochromes P450. After identification of
Edwards et al. [12] and Alterman et al. [13], antibodies spots on 2DE by MALDITOF/MS Galeva and
to cytochromes P450 may be generated using synthetic Alterman [16] concluded that despite of high resolu
peptides: varying their sequences it is possible to gen tion capacity of 2DE the method of 1DE is more rel
erate antibodies to particular P450 isoforms. For anti evant for analysis of cytochromes P450 because of
body generation Edwards et al. [12] used peptides cor their high hydrophobicity and aggregability during the
responding to fourfive Cterminal residues of cyto first stage of 2DE, electrofocusing [16–19]. Analysis
chrome P450. Resultant antibodies demonstrated of mouse liver microsomes by 1DE resulted in identi
high specificity for determination of the cytochrome fication of CYP 3A1, 2C11, 2D2, 2D5, 2A1, 2B1 and
P450 superfamily in the microsomal fraction or liver 2B2 both in control samples and after Phenobarbital
homogenate. However, the antibodies exhibited cross induction, while using 2DE researchers found only
reactivity towards proteins with proteins with identical isoforms 2B1 and 2B2 after Phenobarbital induction
Cterminal fragments, for example, for enzymes [16].

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Petushkova et al. [17, 18] came to the same conclu protein identification, while now priority is given to
sion. These authors optimized conditions of 1DE and quantitative estimations. The quantitative analysis of
determined localization in gel of various CYP isoforms proteins in biological objects represents a nontrivial
determined by MALDITOF/MS. They performed a task due to complexity of the proteomic analysis itself
comparative study of CYP profiling and catalytic because concentrations of thousands of proteins,
activity in liver microsomes from patients with colon which have to be identified and quantified during such
cancer and patients without this pathology. Two proteomic analysis, may differ by several orders of
dimensional electrophoregrams did not reveal spots of magnitude [15]. However, quantitative evaluation of
cytochromes P450, while use of 1DE in combination proteins (including cytochromes P450) is now needed
with MALDITOF/MS resulted in identification of for modeling of processes occurring in the body, for
11 CYP isoforms (2A6, 2C8, 2C9, 2C10, 2D6, 2E1, elucidation of links between phenotype and genotype,
3A4, 4F2, 1B1, 4A11, and 1A2). These authors local and for development of new drugs as well.
ized the region containing cytochromes P450: it is It should be noted that high homology of cyto
located between carboxyesterase (62.5 kDa) and actin chromes P450 observed not only within one family but
(41.0 kDa) [17]. also among various families significantly complicates
After protein separation by 1DE samples were development of methods applicable for isoformspe
subjected to enzymatic hydrolysis and resultant pep cific MS analysis of these proteins [22]. Nevertheless,
tides were analyzed by reverse phase liquid chroma MS capacities for quantitative analysis of cytochromes
tography with MS detection (RPLCESIMS/MS) P450 have clear perspectives because using targeted
[15]. methods it is possible to detect isoformspecific pep
tides insignificantly differed by their primary struc
Sample processing without electrophoresis begins tures as even a single amino acid substitution is suffi
with enzymatic hydrolysis; resultant peptides are sub cient for differential analysis [22].
sequently separated by means of column chromatog
raphy [15]. For example, the first stage of such separa
tion may include column ion exchange chromatogra 2.2.1. Identification of Cytochromes P450
phy followed by subsequent analysis by RPLC with in Biological Objects
MS detection (twodimensional chromatography 2D
LC/MS). Improvement of the resolution technology First studies on cytochrome P450 MS were mainly
with the corresponding increase in the number of focused on protein identification in biological objects.
identified proteins in the first dimension of protein For example, Petushkova et al. used MALDI
separation may be achieved using gel filtration, iso TOF/MS for analysis of microsomal cytochromes
electrofocusing, and reverse phase liquid chromatog P450 in liver specimens from patients with colon can
raphy of native proteins. Thus, using combination cer and from patients without such pathology [17].
with RPLCESIMS/MS it is possible to perform The authors identified 11 isoforms of cytochrome
threedimensional (3D LC) separation of proteins P450: 1A2, 1B1, 2A6, 2C8, 2C9, 2C10, 2D6, 2E1,
and peptides; using this approach authors identified 3A4, 4A11, 4F2. Interestingly, CYP1B1 and CYP4A11
more than 10000 proteins in one experiment [15]. were found only in specimens from cancer patients,
while CYP1A2 was found only in control specimens.
Zgoda et al. [21] compared electrophoretic and Comparison of cytochrome P450 activity assayed with
chromatographic methods used for fractionation of marker substrates and results of the proteomic study
membrane proteins and their peptides during pro revealed a 5⎯10fold decrease of the enzymatic activ
teomic studies. Authors considered results of MS anal ity with 7ethoxy and 7methoxyresorufin as sub
ysis of mouse liver microsomal preparations after strates in tumor specimens, while the MS signal
1DE, twodimensional and threedimensional liquid obtained from these proteins did not reach the level
chromatography (2D LC and 3D LC, respectively). required for reliable identification [17].
The largest number of identified proteins including Using MALDITOF/MS for proteomic analysis of
the highest spectrum of identified isoforms of cyto cytochromes P450 it was possible to detect selectively
chromes P450 in the analyzed samples was obtained CYP4A1, CYP4A3, CYP2A1, CYP2B1, CYP2B2,
using 3D LC with separation of native proteins on a CYP2C11, CYP2D2, and CYP2D5 in rabbit and rat
mRPC18 column as the first dimension followed by liver samples [23]. Liver microsomal proteins obtained
subsequent separation of peptides obtained after enzy after induction of cytochromes P450 in rats by phe
matic proteolysis of these proteins by ion exchange nobarbital or clofibrate administration were initially
and reverse phase chromatography [21]. analyzed by 1DE; the gel region containing proteins
ranged from 45 to 60 kDa was chosen for subsequent
2.2. MassSpectrometry Analysis of Cytochromes P450 trypsinolysis and MS analysis. Using MALDI
TOF/MS for analysis of resultant peptides authors
Mass spectrometry is the most informative tool for [23] differentiated CYP2B1 and CYP2B2 character
cytochrome P450 analysis in biological objects [15, ized by high homology (97%) and differed by only 14
22]. In early proteomic studies MS was used mainly for of 491 residues [23]. In addition to these cytochrome

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 MODERN METHODS OF CYTOCHROME P450 ANALYSIS 127

P450 isoforms CYP1A1, CYP1A2, CYP2B4, lute concentrations of proteins (AQUA, absolute
CYP4B1, and CYP2A10 were found in liver quantification) [31].
microsomes from rabbits after induction with Phe Methods based on chemical modification of pro
nobarbital [23]. teins or their peptides are more popular [15, 22, 28,
Reversephase liquid chromatography combined 29]. They frequently employ derivatizing agents that
with tandem MS and electrospray ionization (RPLC exist in two forms, heavy or light ones (in dependence
ESIMS/MS) is a more reliable method used for cyto of presence of heavy or light isotopes in their struc
chrome P450 profiling [15, 21]. In some studies cyto tures). For comparison of two specimens protein
chrome P450 identification was performed during amino acids are chemically modified by an agent con
general proteomic analysis [24⎯26]. For example, taining either light or heavy stable isotopes. After the
proteomic analysis of liver endoplasmic reticulum in reaction of chemical labeling specimens are mixed and
rats with aflatoxin B1induced tumor revealed analyzed together. Comparison of MS signals from
decreased expression of cytochromes P450 compared peptides modified with heavy and light isotope labels is
with control samples [24]. 2DLC methods were used used for quantification. Using internal standards this
for analysis of the phenobarbital effect on cytochrome approach is also applicable for absolute quantitative
P450 induction in rat liver [25, 26]. Authors found the analysis of proteins in the analyzed specimens with
total increase in cytochrome P450 content and the heavy labels [15, 22, 28, 29].
highest effect was observed in the case of CYP2B2 Such derivatization reactions used for administra
[26]. Nisar et al. used RPLCESIMS/MS for identi tion of isotope labels include acylation of lysine resi
fication of 24 isoforms of rat liver cytochromes P450 dues by deuterated acetic anhydride [35], guanidina
[27]. They found genderspecific expression of some tion and Nterminal group and lysine amino groups
enzymes. For example, CYP2C11, CYP2C22, 2C13, [36, 37], and methylation of peptide amino groups
and CYP2D5 were found only in male rats, while [38, 39]. In addition, there are commercially available
CYP2C12, CYP2C23, CYP2C24, and CYP2D4 were derivatizing agents such as ICAT (“IsotopeCoded
detected only in female rats. Affinity Tag”), an improved cleavable variant of
cICAT (Cleavable Isotope Coding Affinity Tag), ICPL
(isotopecoded protein label) and some others
2.2.2 Methods of Quantitative MS Analysis [40⎯43].
In addition to identification of cytochromes P450 The method of iTRAQ (isobaric Tagging Reagents
in biological objects much attention is now paid to Aminoreactive Quantification) has been developed
their quantitative analysis. Quantitative proteomic for quantitative analysis at the level of fragment ions.
analysis involves two approaches: the first one is based The isotope tag binds to the Nterminal site of a
on comparison of control and experimental states of a polypeptide chain or side εamino groups of lysine. It
biological system of interest and results in relative consists of two groups, a balancer and a reporter,
quantitative evaluation of changes induced by some which represent Npiperazine and carbonyl groups,
treatments [15]. respectively [44]. Derivatizing reagents can vary
Relative quantification may be achieved by direct masses of the balancer from 114 to 117 Da and the
comparison of MS signal intensity (areas under chro reporter from 28 to 31 Da by varying 13C, 15N, and 18O
matographical peaks) or the number of tandem MS content, but their sum remains constant. Tagged pep
obtained from various samples (the method of spectral tides are eluted simultaneously during chromato
counting). This quantitative analysis is known in pro graphic separation, produce the same m/z values on
teomics as Label free quantification [15, 22, 28, 29]. the mass spectrum, but differ by fragmentation spec
tra, where reporter ions provide signals with m/z values
The other approach is based on administration of of 114, 115, 116, and 117 Da for four isobaric tags,
stable isotopes into analyzed proteins (peptides); these respectively. In the fragmentation spectrum the ratio
isotopes may be included into the backbone amino of the ion signals with m/z values of 114, 115, 116, and
acid or into their side groups [15, 22, 28, 29]. Various 117 correspond to the ratio of protein concentrations
methods exist for administration of stable isotopes into in specimens [45, 46].
proteins [28]. For example, proteins may be labeled One of the earliest methods of stable isotope
during their translation in cells incubated in a medium
administration used H216O and H218O during enzy
containing stable isotopes 13C and/or 15N. This
method of protein labeling is known as SILAC (Stable matic hydrolysis of proteins [47⎯55]. The protocol
Isotope Labeling with Amino acids in Cell culture) used by Fenselau et al. [48] included trypsinolysis of
[30]. In the case of higher organisms isotopelabeled one sample in the buffer prepared in H216O, while
amino acids may be delivered with foodstuff; however, trypsinolysis of another sample was carried out in the
this method is very expensive and inapplicable for buffer prepared using H218O. Before analysis the sam
human specimens [32⎯34]. Individual peptides con ples were pooled. The reaction of 18O/16O isotope
taining isotopelabeled amino acids may be synthe labeling yields Cterminal labeled tryptic peptides.
sized chemically and used for measurements of abso Results are evaluated using protocols described in

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128 MOSKALEVA, ZGODA

details by RamosFernandez et al. [49] and Lopez tions. According to these calculations, CYP1A2,
Ferrer et al. [50]. CYP2A6, CYP2C9, CYP2E1, and CYP3A4 belong to
the group of high concentrations and each form repre
sent about 10% of total CYP content; the group of
2.2.3. LabelFree Relative Quantitative MS moderate concentrations includes CYP2C8,
Determination of Cytochromes P450 CYP4A11, and CYP4F2 (0.5⎯1%), while the group of
Alterman et al. provided a good example of label cytochromes P450 of low concentrations (<0.5%)
free quantitative MS determination of cytochromes includes CYP2B2, CYP2J2, CYP4F11, CYP4F12,
P450 by MALDITOF/MS [56]. Two isoforms of CYP4V2, CYP7B1, CYP8B1, CYP20A1, and
mouse cytochrome P450 (CYP2B1 and CYP2B2) and CYP51A1. However, authors [58] indicate that
three isoforms of human cytochromes P450 emPAIbased quantitative evaluations strongly
(CYP1A2, CYP2E1, and CYP2C19) were quantita depend on methods and analytical equipment used for
tively determined using a single isoformspecific pep analysis [58]. For example, CYP3A4 was analyzed by
tide synthesized for each protein. The method tested three methods. The ion trap MS analysis of samples
using recombinant cytochromes P450 gave higher val after 1DE revealed that CYP3A4 represented 28.6%
ues than CO spectrophotometry, which determines of total CYP content. The QTOF analysis of samples
only the content of the active form of the enzyme. after 1DE revealed that CYP3A4 represented 10.4%
Methods based on chromatographic separation of of total CYP content, while after 2DLC the QTOF
sample components in combination with mass spec analysis yielded the value of 16.4%. More accurate
trometry and electrospray ionization (RPLCESI results were obtained during targeted quantitative
MS/MS) appear to be more informative. Duan et al. analysis using a triple quadrupole (QQQ) mass spec
[57] proposed the method of quantitative determina trometer and isotopelabeled peptides as a standard.
tion of human CYP2D6, CYP2C9, CYP2C19, For example, using synthetic proteotypic peptides
CYP2E1, and CYP3A4; it is based on chlorination of authors [58] determined that concentrations of
cysteine residues in analyzed samples and bromina CYP2E1 and CYP1A2 in three specimens of human
tion in the standard. In the range of concentration microsomes were in the ranges 88–200 and 163–263
from 10 fmol/μg to 5 pmol/μg the experimental error pmol/mg of protein, respectively.
of determination of human cytochromes CYP2D6,
CYP2C9, CYP2C19, CYP2E1, and CYP3A4 added to 2.2.4. IsotopeBased Methods of MS Analysis
the mouse liver microsome matrix was less than 20%. of Cytochromes P450
However, authors [57] did not describe analysis of real
samples. For relative quantitative analysis of cytochromes
Seibert et al. [58] used the method of relative quan P450 Jia et al. [35] used acetylation of peptides by
titative evaluation of cytochromes P450 in human Nterminal amino groups using D6acetyc anhydride
microsomes simultaneously with their identification followed by high resolution MS analysis. Studying the
during general proteomic study. These authors com effect of carbon tetrachloride on CYP expression in rat
pared exponentially modified protein abundance liver the authors identified 17 CYP isoforms; concen
indexes (emPAI), which denote the ratio of observed trations of CYP2C11, CYP3A2, and CYP2E1 demon
to observable peptides that could be potentially strated more than 2fold decrease in response to car
detected to a given protein. bon tetrachloride treatment, while concentrations of
CYP2C6, CYP2B2, and CYP2B1 increased by 1.7,
PAI = Ndetected/Ntheor, 3.9, and 4fold, respectively. Detailed results of this
where Ndetected is the number of peptides detected for study are summarized in Table 1.
particular cytochrome P450 analyzed during the pro Huang et al. [59] investigated gender differences in
teomic study and Ntheor is the theoretically possible rat liver content of cytochromes P450. The relative
number of peptides. quantitative analysis was performed using LCMS; the
isotope label was administered during sample methy
emPAI = 10PAI – 1. lation using D or Hformaldehyde. Proteomic analy
Relative content of a particular isoform was evalu sis resulted in identification of 21 CYP isoforms;
ated as a part of total cytochrome P450 content using CYP2C11, CYP2C13, CYP2B3, CYP2C70, and
the following formula: CYP3A2 were more specific for males, while CYP2A1,
CYP2C7, and CYP2D26 were specific for female rats.
content (%) = [(emPAI Mr)/Σ [(emPAI Mr)] × 100,
Jenkins et al. [42] used a cysteine specific ICAT
where Mr is molecular mass of the analyzed isoform, reagent for relative and absolute quantitative analyses of liver
Σ[(emPAI Mr)] is the sumproduct of emPAI and Mr microsomal cytochromes P450 in control mice and after
for all identified CYP isoforms. treatment by phenobarbital or methylcholanthrene.
Based on results of experimental quantification Using this quantitative approach authors identified three
identified CYP isoforms have been subdivided into groups of isoforms belonging to the 2C subfamily
three groups of high, moderate, and low concentra (CYP2C29/CYP2C37/CYP2C50, CYP2C38/CYP2C39,

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 MODERN METHODS OF CYTOCHROME P450 ANALYSIS 129

and CYP2C40) and found different responses of Table 1. Relative changes in the content of liver microsomal
CYP2C29, CYP2C40, and CYP2C50 to the chemical cytochromes P450 (CYPs) in rats treated with carbon tetra
inducers. In contrast to CYP2C40, and CYP2C50 phe chloride (modified from [35])
nobarbital administration caused a 2⎯3fold increase in Ratio of peptide intensity
CYP2C29/39 expression, while isoforms of the CYP2B Identified CYP isoform in carbon tetrachloride treated
subfamily demonstrated a 4⎯13fold increase in their versus control sample
expression. In addition, methylcholanthrene was a more
potent inducer of CYP1A1 expression compared with 2C11 0.34 ± 0.03
CYP1A2. Since quantitative evaluation by ICAT is based 3A2 0.35 ± 0.02
on cysteine containing peptides Jenkins et al. indicate 2E1 0.55 ± 0.04
inapplicability of this approach for differentiation of
members of the 3A, 2B, and 2D subfamilies because of 2C6 1.74 ± 0.02
identify of their cysteine containing peptides. It also 2B2 3.88 ± 0.47
should be noted that in most CYP the number of cysteine 2B1 4.07 ± 0.06
residues does not exceed four and they are usually located 2D26 1.06 ± 0.02
within conservative sites of amino acid sequences and this
also limits ICAT applicability for CYP analyses. 2C13 0.86 ± 0.11
For absolute quantification of CYPs Jenkins et al. 2D10 1.47 ± 0.02
[42] synthesized peptides of 16 individual isoforms of 2C7 1.15 ± 0.05
the CYP subfamilies 1A, 1B, 2A, 2B, 2C, 2D, 2E, 2F, 2A1 1.04 ± 0.07
2J, 3A, and 4A. The determined concentrations of 2C23 1.15 ± 0.33
these proteins were in the range of 10⎯100 fmol/μg of
2A3 0.99 ± 0.03
protein. The authors indicated that the method of
absolute quantitative analysis provided higher sensitiv 1A2 0.76 ± 0.09
ity and accuracy of measurements. For example, the 4A2 1.20 ± 0.29
reported concentrations cytochromes of the 4A sub 2C12 1.62 ± 0.01
family (2.71 fmol/mg) were detected only during tar
2C70 0.66 ± 0.06
geted absolute quantitative analysis [42]. Table 2 shows
concentrations of CYP isoforms in control samples
and after induction by phenobarbital (concentrations
of CYP isoforms significantly influenced by phenobar labeled peptide GTVVVPT(*L)DSVLYDNQEFPD
bital treatment are shown in bold). PEK (the *L residue was labeled by six 13C and one
15N) as an internal standard. This peptide is unique for
Wang et al. [60] used the iTRAQ (isobaric Tagging the human CYP2E1. Subsequent analysis of this sam
Reagents Aminoreactive Quantification) method for ple by QQQ revealed the CYP2E1 content of
relative quantitative analysis of CYPs in human 100 fmol/μg of total protein.
healthy liver samples and the liver carcinoma tissue.
Equivalent (by total protein content) amounts of Langenfeld et al. used the AQUA method with iso
microsomal samples of healthy and the liver carci topelabeled synthetic peptides as standards for quan
noma tissue were subjected to enzymatic cleavage and titative determination of CYP2D6 by means of QQQ
the resultant peptides were modified by variants of the [63]. In 30 specimens of human liver microsomes
isobaric tag (iTRAQ114 and iTRAQ117). Analysis CYP2D6 content varied from 0.8 to 81 fmol/μg of
of the pooled samples by 2DLCMS/MS revealed microsomal protein and in specimens from individuals
decreased content of CYP2D6 in the carcinoma tissue with two nil alleles (*3, *4, *5; PM genotype) its con
versus healthy tissue samples. tent was negligible. These results well correlated with
Lane et al. [61] described the method of stable iso enzyme activity determined by dextromethorphan.
tope administration during enzymatic protein cleav
Wang et al. [14] performed quantitative analysis of
age by means of H216O and H218O for liver CYP profil CYP3A4 and CYP3A5 in human microsomes. Pep
ing in mice treated with 1,4bis[2(3,5dichloropy tides obtained after trypsinolysis of recombinants
ridyloxy]benzene (TCPOBOP). Authors identified CYPs were used as standards, and corresponding iso
17 CYP isoforms and 16 isoforms were quantitatively topelabeled synthetic peptides were used as internal
analyzed. Using this method it was possible to differ standards for control of reproducibility of the method
entiate such isoenzymes as CYP2B10 and CYP2B20 and ionization efficiency. The resultant mean values of
(which share 87% identity in their amino acid CYP3A4 and CYP3A5 content were 67 and
sequences), CYP2C29, CYP2C37, and CYP2C38 4 pmol/mg of microsomal protein, respectively. The
(71% identity in their amino acid sequences). individual variability somewhat lower than two orders
Wang et al. [62] performed absolute quantification of magnitude for CYP3A5 (from 0.3 to 20 fmol/μg)
of CYP2E1 in human liver specimens by means of a and about 30 times for CYP3A4 (from 9 to
single isoformspecific peptide and the isotope 322 fmol/μg).

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130 MOSKALEVA, ZGODA

Table 2. The effect of Phenobarbital induction on concentrations of cytochromes P450 (modified from [42])
Concentration in control, After Phenobarbital induction,
P450 isoform Peptide
fmol/μg of protein fmol/μg of protein
1A1 CIGETIGR 5.38 ± 0.35 5.21 ± 1.14
1A2 CIGEIPAK 1.38 ± 0.24 1.25 ± 0.18
1B1 CIGEELSK 14.11 ± 1.01 14.99 ± 0.51
2A4 YCFGEGLAR 11.53 ± 1.22 13.02 ± 0.26
2A12 FCLGDSLAK 15.07 ± 1.62 14.99 ± 1.32
2B9/10/13/20 ICLGESIAR 11.41 ± 195 68.97 ± 5.24
2C29/37/50 ICAGEGLAR 55.84 ± 403 171.18 ± 23.03
2C38/38 VCAGEGLAR 7.58 ± 1.09 7.48 ± 0.96
2C40 ICVGESLAR 16.15 ± 1.93 15.85 ± 1.97
2D9/11 SCLGEALAR 12.42 ± 0.70 7.56 ± 1.39
2D10/22/26 SCLGEPLAR 21.68 ± 1.05 19.61 ± 0.42
2E1 VCVGEGLAR 35.13 ± 1.58 30.38 ± 2.78
2F2 LCLGEPLAR 21.74 ± 3.34 3.34 ± 16.27
2J5 ACLGEQLAK 9.05 ± 0.94 8.82 ± 0.62
3A11/13/16 NCLGMR 5.48 ± 0.63 19.56 ± 1.16
4A10/11/12/14 NCIGK 2.71 ± 0.86 4.32 ± 0.93

Kawakami et al. [64] used the AQUA method for that can be used for evaluation of functional and
MS determination of 11 isoforms of human CYPs. detoxification function of the liver in medical practice
Authors indicated difficulties in selection of proteo or prediction of putative toxic effect of drugs. CYP
typic peptides for the quantitative analysis due to high activity is evaluated by concentration of a forming
homology of the isoforms. Peptides had to lack chem marker metabolite. According to literature data, the
ically labile amino acids, missed sites of trypsinolysis methods used for determination of products of the
and known posttranslational modifications. A prelim
enzymatic reaction include HPLC with UV or MS
inary total proteomic study of the sample did not
reveal such peptides for all isoforms of interest and so detection, radioactivity determination, fluorescence
authors synthesized theoretical peptides; their or luminescence [65].
response was confirmed during targeted proteomic
analysis.
For each protein authors synthesized one isotope 3.1. Methods Based on Determination of Radioactivity
labeled peptide and after preliminary calibration they
determined concentrations of the analyzed CYPs in These methods are based on 3H or 14Clabeled
10 specimens of human microsomes. Five isoforms, substrates, which are selectively metabolized by inves
CYP2C9, CYP2E1, CYP1A2, CYP2C8, and tigated CYP isoforms [65⎯67]. After sample separa
CYP3A4, were classified as high copied; their content tion by HPLC metabolite concentrations are deter
exceeded 8 fmol/μg of microsomal protein. mined by means of a radiochemical detector. In some
CYP2C19, CYP3A5, CYP3A43, and CYP2B6 were cases enzymatic reaction results in release of 14C
classified as low copied ones (their content was below formaldehyde, which may be extracted from the reac
2 fmol/μg. The content of CYP2A6 and CYP2D6 was tion mixture and detected by means of a scintillation
in the range 2⎯8 fmol/μg of microsomal protein. counter. Some versions of this method do not require
Interestingly, the expression levels were characterized chromatographic separation; they are realized as scin
by demonstrated 10⎯20fold variations in individual tillation beads impregnated with polyethyleneimine,
specimens [64].
which can bind 14Cformaldehyde. Formation of 14C
formaldehyde results in scintillant excitation and
3. PHENOTYPING AS THE MODE emission [67]. These methods have been proposed for
FOR EVALUATION OF THE FUNCTIONAL evaluation of CYP inhibition by novel drug substances;
STATE OF THE MONOOXYGENASE SYSTEM their advantage consists in used of any substrate
The functional capacity of the monooxygenase sys (including endogenous compounds) for evaluation of
tem for substrate oxidation is an important parameter the enzymatic activity.

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 MODERN METHODS OF CYTOCHROME P450 ANALYSIS 131

3.2. Bioluminescent Methods complex biological systems such as microsomes,

This technology is based on the use of substrates hepatocytes, cell lines, and tissue sections [65, 69, 70].
that release luciferin during their CYPdependent oxi Enzymatic activity of various CYP isoforms is eval
dation (the commercial product of Promega, USA). uated by products formed during incubation with sub
Incubation of the monooxygenase system containing strate and detection is usually performed using a triple
CYP in the presence of the luminogenic substrate is quadrupole (QQQ) mass spectrometer [74⎯87]. QQQ
accompanied by luciferin release and appearance of consists of two quadrupoles and the collision cell is
luminescence, which is proportional to CYP activity. located between them. The first quadrupole allows to
Since such substrates are not very isoform specific it is pass only parent ions of a targeted substance, which
better to use this method in reconstituted monooxyge forms characteristic daughter ions in the collision cell;
nase systems containing particular recombinant or these ions enter the second quadrupole. The presence
isolated enzymes rather than in experiments with liver of two filters allows to obtain a selective signal of the
microsomal preparations [65]. This method is mainly analyzed substance and to increase determination
used for evaluation of the effects of novel drug sub sensitivity. Table 3 briefly summarizes results of analy
stances on CYP activity. sis of literature data on the most frequently used CYP
substrates and MS parameters used for detection
[74⎯87].
3.3. Fluorescence Methods
For example, authors [74–82] used phenacetin as
The fluorescence method is the most frequently the substrate for analysis of CYP1A2 activity and ace
used method for evaluation of CYP activity. It is based taminophen, its reaction product. After CYP incuba
on the use of substrates which are metabolized by tion with phenacetin samples were analyzed by reverse
CYPs with formation of fluorescent products. phase liquid chromatography with electrospray ion
Changes in the fluorescence intensity during CYP ization and MS detection [74–82]. The [M + H]+ ion
incubation with corresponding substrate is the quanti with m/z of 152 Da (corresponding to the protonated
tative measure of the fluorescent product formed and form of phenacetin) and the ion fragment with m/z of
therefore the measure of enzyme activity. This is the 110 Da were used in QQQ as the parent and daughter
simplest, express, and readily reproducible method ions, respectively. Reliability of this identification was
and so most pharmaceutical companies use this controlled by coincidence with retention time of the
approach for analysis of CYP inhibition of novel drug sample and the standard substance.
substances [65, 68–73]. BD Bioscience (USA) pro
duces special kits for such screening. However, appli
cability of this method is limited by low isoform spec 4. COMPARISON OF CATALYTIC ACTIVITY
ificity of the substrate used and so it is mainly applica OF CYTOCHROMES P450 WITH THEIR
ble for studies of CYP inhibition using recombinant CONTENT
enzymes. For example, Yan et al. [69] investigated
interaction of nine of the most common substrates Comparison of catalytic activity of the monooxy
used for fluorometric assay with 29 recombinant CYP genase system with quantitative content of pharmaco
isoforms. CYP1A1 and CYP1B1 were characterized logically important CYP isoforms and also changes in
by comparable Km values for resorufin benzyl ether; these parameters caused by inducers and inhibitors are
the most interesting in terms of modeling of drug
CYP1A1 and CYP3A4 had comparable Km values for effects.
dibenzyl fluorescein. In the BD Bioscience kits 3flu
oro7methoxycoumarin is used for determination of Results of MS analysis of CYP2D6 have been com
CYP2E1 and CYP2C9 activity, while 3cyano2 pared with results of determination of CYP activity by
ethoxycoumarin is used for determination of CYP1A2 the rate of dextromethorphan hydroxylation [63].
and CYP2C19 activity. Studies were carried out using a genotyped set of
30 specimens of human microsomes and authors
found a correlation (r = 0.8) between activity and con
3.4. Methods Based on HPLC with UV centration of CYP2D6. The authors believe that eval
and MS Detection uation of the functional activity of CYP2D6 would be
Methods based on HPLC determination of reac useful for prevention of side reactions.
tion products appear to be the most selective and accu In human liver microsomes other researchers [14]
rate in studies of CYP in various biological objects determined concentrations of CYP3A4 and CYP3A5
such as microsomes or tissue sections. Earlier detec by means of RPLCESIMS/MS and Western blot
tion was performed using an UV detector; now meth analysis and their catalytic activity towards testoster
ods employing MS detection become more and more one (CYP3AX), midazolam (CYP3AX), itraconazole
popular [65, 69, 70]. In such studies drugs substances (CYP3A4), and vincristine (CYP3A5). They demon
selectively metabolized by certain CYP isoforms are strated that the rates of itraconazole hydroxylation
used as substrates. Using isoformspecific substrates it mainly catalyzed by CYP3A5 characterized by high
is possible to evaluate activity of certain enzymes in correlation (0.97) with enzyme concentration deter

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132 MOSKALEVA, ZGODA

Table 3. Substrates used for assay of activities of pharmacologically important cytochrome P450 isoforms by means of MS
determination of concentrations of enzymatic oxidation products (summarized from [74⎯87])
Analyzed Parent and daughter ions used during QQQbased
Marker substrate Measurable metabolite
isoform detection
1A2 Phenacetin Acetaminophen 152 → 110 [74⎯82]
Melatonin 6Hydroxymelatonin 249 → 190 [83]
Ethoxyresorufin Resorufin 214 [85]
2A6 Coumarin 7Hydroxycoumarin 161 → 133 Neg [75, 80]
163 → 107 [83 85] 163 [84]
2B6 Bupropion 2Hydroxybupropion 256 → 139 [74, 80]
256 → 238 [83, 84]
256 → 184 [86]
2D6 Dextromethorphan Dextrorphan 258 → 201 [74, 79, 82, 87]
258 → 157 [77, 80, 81]
258 → 199 [83]
Bufuralol 1Hydroxybufuralol 278 → 187 [75, 78, 85]
2E1 Chlorzoxazone 6Hydroxychlorzoxazone 184 → 120 Neg [77, 79, 80, 83]
2C8 Amodiaquine Desethyl amodiaquine 328 → 283 [74, 80, 83, 86]
Paxitaxel 6αHydroxypaxitaxel 870 → 286 [85]
2C9 Diclofenac 4Hydroxydiclofenac 312 → 231 [71, 75, 77, 85]
312 → 266 [76, 81]
310–266 Neg [83]
Tolbutamide 4Hydroxytolbutamide 287 → 188 [74, 82]
287 → 89 [78]
287 → 171 [80, 83]
285 → 186 Neg [79]
2C19 Mephenytoin 4Hydroxymephenytoin 235 → 186 [75]
235 → 150 [76, 77, 81, 82, 85]
235 → 133 [79]
233 → 190 Neg [80]
Omeprazole 5Hydroxyomeprazole 362 → 214 [74, 78, 83]
Omeprazole Desmethyl omeprazole 332 → 198 [83, 84]
3A4,5 Midazolam 1Hydroxymidazolam 342 → 324 [75, 80, 83, 85, 87]
342 → 203 [71, 76, 77, 79, 86]
342 → 297 [78]
326 → 391 [81]
Testosterone 6Hydroxytestosterone 305 → 287 [74, 82]
305 → 269 [76, 80, 81, 83]
3A4,5 Omeprazole Omeprazole sulfone 362–150 [83, 84]
Omeprazole 3Hydroxyomeprazole 362–214 [83, 84]
Dextromethorphan 3Methoxymorphinan 258 → 213 [79]
Felodipine Dihydrofelodipine 382 → 354 [80]

mined by MS. Interestingly, correlations between pro CYP3A5) in the culture of human hepatocytes after
tein concentrations determined by MS and corre induction by methylcholanthrene, rifampicin, and
sponding catalytic activities were found for all ana phenobarbital and in control cells. The analysis was
lyzed enzymes (correlation coefficient exceeding 0.8), carried out by the MRM method in the presence of
while results of immunoenzyme assay did not demon isotopelabeled peptides selected for each protein
strate such correlation with MS data. (three peptides per protein). Authors indicate that the
Williamson et al. [88] performed relative quantita culture of human hepatocytes is a valuable model
tive analysis of the pharmacologically important CYP object for testing of novel drugs. Authors compared
isoforms (CYP1A2, CYP2B6, CYP3A4, and enzyme activity determined with specific substrates,

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 MODERN METHODS OF CYTOCHROME P450 ANALYSIS 133

data of mRNA analysis and MS evaluation of the pro 8. Raunio, H., Hakkola, J., Hukkanen, J., Pelkonen, O.,
tein content. The most potent induction was noted in Edwards, R., Boobis, A., and Anttila, S., Arch. Toxicol.
the case of CYP1A2 after treatment of human hepato Suppl., 1998, vol. 20, pp. 465⎯469.
cytes with methylcholanthrene. Interestingly, the 9. Luss, H., Li, R.K., Shapiro, R.A., Tzeng, E.,
methylcholanthreneinduced changes demonstrated McGowan, F.X., Yoneyama, T., Hatakeyama, K.,
the 141fold increase in mRNA level (coding this pro Geller, D.A., Mickle, D.A., Simmons, R.L., and Bil
tein), the 60fold increase in the catalytic activity of liar, T.R., J. Mol. Cell Cardiol., 1997, vol. 29, pp. 1153⎯
1165.
this protein and the 45fold increase in the protein
content evaluated by quantitative MS analysis. Higher 10. PradetBalade, B., Boulme, F., Beug, H.,
Müllner, E.W., and GarciaSanz, J.A., Trends Biochem.
correlations were found for other isoforms. For exam Sci., 2001, vol. 26, pp. 225⎯229.
ple, the increase in CYP3A4 content observed
11. Caron, E., Rioux, N., Olivier, N., LebelTalbot, H.,
after induction by phenobarbital authors observed the and Hamelin, B., J. Biochem. Molecular. Toxicology,
14fold increase in CYP3A4 mRNA, the 23fold 2005, vol. 19, pp. 368⎯378.
increase in the enzyme activity, and the 16fold 12. Edwards, R.J., Boobis, A.R., and Davis, D.S., Drug
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Possible lack of correlations between mRNA content
13. Kornilayev, B.A. and Alterman, M.A., Toxicology in
and protein content of corresponding CYP is attrib Vitro, 2008, vol. 22, pp. 779–787.
uted by posttranslational and transcriptional regula
14. Wang, M.Z., Wu, J.Q., Dennison, J.B., Briges, A.S.,
tory mechanisms of protein content. Authors indicate Hall, S.D., Kornbluth, S., Tidwell, R.R., Smith, P.C.,
that there is much interest in elucidation of interrela Voyksner, R.D., Paine, M.F., and Hall, J.E., Proteom
tionships between quantitative characteristics of CYP ics, 2008, vol. 8, pp. 4186–4196.
and their activity [88]. 15. Walter, T. and Mann, M., J. Cell Biol., 2010, vol. 190,
pp. 491⎯500.
CONCLUSIONS 16. Galeva, N. and Alterman, M., Proteomics, 2002, vol. 2,
pp. 713–722.
Despite of a significant number of reports on CYPs 17. Petushkova, N.A., Kanaeva, I.P., Lisitsa, A.V., Sher
the development of effective methods of their quanti emetyeva, G.F., Zgoda, V.G., Samenkova, N.F.,
tative analysis still represents an important task. Some Karuzina, I.I., and Archakov, A.I., Toxicology in Vitro,
pharmacologically important CYP isoforms still wait 2006, vol. 20, pp. 966–974.
for better characterization and changes in CYP con 18. Petushkova, N.A. and Lisitsa, A.V., Methods Mol. Biol.,
centrations during inhibition or induction of these 2012, vol. 909, pp. 63⎯82.
enzymes require more accurate determination. Since 19. Sutton, C.W., Sutherland, M., Shnyder, S., and Patter
the monooxygenase system and especially CYPs play a son, L.H., Proteomics, 2010, vol. 10, pp. 327–331.
key role in metabolism of xenobiotics studies of inter 20. Makino, T., Kinoshita, J., Arakawa, S., Ito, K.,
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ble inhibitory and inducer effects is an obligate stage of and Nakayama, H., J. Toxicol. Sci., 2009, vol. 34,
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