the united states food 
 and drug administration and european medicines 
 agency advise that , to improve safety in clinical development and 
 postapproval , the potential for a new therapeutic agent to interact 
 with established medications ( drug  drug interaction , ddi ) should 
 be assessed as early as possible during preclinical development .
one major mechanism of ddi is the ability of chemical agents to 
 regulate the expression of drug metabolism enzymes and transporters , 
 often through the modulation of nuclear hormone receptor ( nhr ) activity , 
 thereby altering the efficacy of both themselves and other compounds . 

it is valuable , therefore , to have a comprehensive understanding of 
 the levels of expression of these protein factors and how they are 
 modulated during therapy . in the liver , the primary site of 
 drug metabolism ,
the majority 
 of phase i ( modification ) reactions are carried out by cytochrome 
 p450 ( cyp ) , with additional contributions made by alcohol dehydrogenase 
 ( adh ) , aldehyde dehydrogenase ( aldh ) , aldo - keto reductase ( akr ) , epoxide 
 hydrolase ( ephx ) , and flavin - containing monooxygenase ( fmo ) superfamilies .
udp glucuronosyltransferases ( ugt ) and glutathione 
 s - transferases ( gst ) enact most phase ii ( conjugation ) events . during laboratory study , if a broad expression 
 profile of these dmes is required , dna microarray or high - density 
 rt - pcr arrays are typically employed . while providing a practicable 
 platform for this type of analysis
a recent study by ohtsuki 
 and colleagues demonstrated a poor correlation between protein and 
 mrna levels for multiple cyp , ugt , and drug transporters , with only 
 a handful of exceptions . in this study , 

the direct measurement of protein expression , as opposed to the measurement 
 of mrna , correlated far better with enzymatic activity and is therefore 
 a more appropriate readout for the evaluation of drug 
western blotting 
 and other antibody - based approaches are the mainstay 
 of protein expression analysis for dme , but our lab and others routinely 
 struggle to interpret data due to the high degree of sequence homology 
 of superfamily members , and hence cross - reactivity of antibody preparations . 
 in order to circumvent this issue , recent developments have been made 
 in stable isotope dilution mass spectrometry - based proteomics to simultaneously 
 detect and quantify cyp and other drug metabolism related proteins .
these studies utilize an absolute quantification technique ( aqua ) 
 where known quantities of multiple synthetic stable isotope peptides 
 for a protein / proteins of interest are spiked into proteolytic protein 
 digests derived from liver samples , prior to lc - ms / ms analysis in 
 a multiple reaction monitoring ( srm ) mode .
 
 unlabeled peptides co - elute , co - ionize , and are only differentiated 
 by the difference in mass through lc - ms / ms analysis . using peak intensity 
 ratios of the pairs of light and heavy isotopes , concentrations of 
 analyte peptides
a modified version of this procedure 
 using stable isotope - labeled proteins expressed in and purified from escherichia coli as internal standards has also been 
 developed which , post - translational modification and extraction variability 
 excepted , accounts for efficiency of enzymatic digestion .
but for comprehensive proteomic analysis , stable isotope labeling 
 by amino acids in cell culture ( silac ) or in whole organisms ( in vivo silac , silam ) permits the simultaneous quantification 
 of thousands of proteins with a much improved confidence due to the 
 fact that both light and heavy analytes share near - identical chemical 
 properties and environment .
label - free shotgun proteomics is an alternative commonly used 
 mass 
 spectrometry based proteomics strategy .
there are two mainstream label - free based lc - ms / ms approaches based 
 on either spectral counting or ion intensity .
the former compares 
 the number of ms2 spectra assigned to a protein between samples , while 
 the latter compares intensities of each precursor ion between samples . 

this approach aligns precursor ions between all lc - ms / ms 
 runs using defined retention time and m / z windows .
the signal intensities in each window for each sample are 
 then integrated , normalized and compared between samples or groups . 

precursor ions showing significant differences or all precursors with 
 ms2 spectra can then be identified .
the success of this approach relies 
 on tight control in sample processing and consistency in retention 
 time in lc separation . in the current study
, we harness the 
 advantages of in vivo silac materials in conjunction 
 with the label - free shotgun proteomics 
 concept to quantify dmes in mouse liver , although the approach could 
 be used for targeted quantification of any detectable protein or protein 
 group of interest . peptides from dmes were identified in a metabolically 
 labeled spike - in standard lysate and a complementary list of unlabeled 
 peptides was constructed .
this list of peptide pairs was stress - tested 
 with a range of heavy to light input sample ratios and only the most 
 reliable were retained .
the final peptide list allowed us to quantify 
 changes in expression of 51 dmes and was used to generate a constitutive 
 androstane receptor ( car ) activation signature .
1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene , 
 3,3,5,5-tetrachloro-1,4-bis(pyridyloxy)benzene ( tcpobop ) , 
 corn oil , dl - dithiothreitol , and iodoacetamide were purchased 
 from sigma ( dorset , uk ) .
all mice were maintained 
 under standard animal house conditions , with free access to food and 
 water , and a 12 h light/12 h dark cycle .
all animal work was carried 
 out on male 8-week - old c57bl/6j mice in accordance with the animal 
 scientific procedures act ( 1986 ) and after local ethical review .
mice 
 were administered either tcpobop ( a single intraperitoneal injection 
 at 3 mg / kg ) in corn oil vehicle at 10 l / g body weight , or the 
 same volume of corn oil vehicle alone .
animals were sacrificed 7 days 
 after dosing , and liver tissue excised and snap - frozen in liquid nitrogen 
 for storage at 80 c . for lc - ms / ms ,
frozen liver tissue 
 was thawed by addition of 9 volumes of sdt lysis buffer ( 4% sds , 0.1 
 m dtt , 100 mm tris - hcl ph7.6 ) then homogenized by rotor - stator ( 2 
  5 s at 20k revolutions ) .
homogenate was heated to 95 c 
 for 5 min , sonicated ( 2  5 s ) , and then centrifuged at 16  000 g for 10 min .
supernatant ( protein sample for analysis ) 
 was removed , aliquoted and stored at 80 c until use . 

protein samples ( total of 30 g / well ) were electrophoresed through 
 a 12% bis - tris gel in mops running buffer supplemented with antioxidant 
 ( all life technologies , paisley , uk ) alongside a spectra multicolour 
 broad range protein ladder ( thermo fisher scientific , waltham , ma ) . 

gels were stained with coomassie blue , destained , and then rehydrated 
 with milli - q water .
gel regions containing proteins of interest , as 
 described in the results section , were removed 
 with a clean scalpel , sliced finely ( ca . 1  1 mm cubes ) , and 
 added to 1.5 ml pcr - clean eppendorf tubes ( eppendorf , hamburg , germany ) . 

in - gel trypsin digest and peptide extraction was carried out according 
 to the method of schevchenko and colleagues .
peptide sample concentration was determined by nanodrop ( thermo 
 fisher scientific ) and adjusted to 0.2 mg / ml in water containing 0.1% 
 ( v / v ) trifluoroacetic acid . for western
briefly , tissue was homogenized in 3 volumes of kcl buffer ( 1.15% 
 w / v potassium chloride , 10 mm potassium phosphate , ph7.4 ) by rotor - stator 
 ( 2  5 s at 20k revolutions ) followed by centrifugation at 11  000 g for 15 min .
supernatant was ultracentrifuged at 100  000 g for 1 h and the resulting pellet resuspended in kcl buffer 
 containing 0.25 m sucrose .
protein concentration was adjusted to 1 
 mg / ml in lds sample buffer ( life technologies ) before electrophoresis 
 through 10% acrylamide gels for 1 h at 200 v , followed by transfer 
 onto nitrocellulose membranes ( 1 h at 100 v ) .
our in - house panel of rabbit 
 polyclonal antibodies for cyp detection has been summarized previously .
fold changes were calculated from chemiluminescent 
 signal intensity on a fujifilm las-3000 imager ( fujifilm , tokyo , japan ) . a nanoflow 
 liquid chromatograph ( agilent 1200 , agilent , santa clara , ca ) with 
 a ltq - orbitrap xl (
approximately 0.4 g total peptide was loaded 
 onto a trap column at a flow rate of 10 l / min for 3 min and 
 the flow was then reversed to an agilent zorbex nano c18 column ( 0.0075 
 mm i d ; 15 cm ; 3 m particle size ) .
the peptides were resolved 
 with a 3 h binary gradient at a flow rate of 300 nl / min as follows : 
 0% buffer b for 5 min followed by 230% buffer b for 140 min , 
 3090% buffer b for 15 min , 900% buffer b for 10 min , 
 and 0% buffer b for 10 min .
buffer a contained 2% acetonitrile and 
 0.1% formic acid in water , and buffer b contained 0.1% formic acid 
 in acetonitrile .
the column was periodically cleaned with a 2 l 
 injection of buffer containing 50% acetonitrile and 0.1% formic acid 
 in water . a proxeon nanospray source with a stainless steel emitter 
 (
thermo fisher scientific ) was used to interface the agilent nanolc 
 and ltq - orbitrap .
the orbitrap was 
 tuned using glu - fibrinogen b peptide . for the protein / peptide identification , 
 a method that consisted of full scans between 330 and 1500 amu ( in 
 orbitrap ) and data dependent
ms / ms with top six precursor ions ( 2 to 4 charged ) in ltq was employed .
orbitrap was 
 operated in a profile mode at the resolution of 30  000 or 60  000 
 with a lock mass set at 445.1200 ( polycyclodimethylsiloxane ) , and ltq was operated in a centroid mode with 
 isolation width = 1 ( m / z ) , normalized 
 collision energy = 0.25 , and activation time = 30 ms .
the max fill 
 times for orbitrap and ltq were set at 500 and 50 ms , respectively . 
 a dynamic exclusion of 30 s was used to maximize the acquisition of 
 ms2 on peptides with lower intensity .
for thr / sim analysis , a method 
 that consisted of full scans between 330 and 1500 a.m.u ( in orbitrap ) 
 and data dependent ms / ms scans with or without defined precursors 
 was employed .
a dynamic exclusion of 30 s and a threshold of 500 counts 
 to trigger ms2 were also applied for ms / ms scans .
nontargeted data 
 dependent ms / ms was performed when there was no targeted precursor 
 found in the ms scan .
further details can be found in the method and 
 tune files ( supporting information files 
 s-1 and s-2 ) .
protein and peptide 
 database search was 
 carried out using peaks version 6 ( bioinformatics solutions , waterloo , 
 canada ) with an ipi - mouse database ( version 3.87 , european bioinformatics 
 institute , hinxton , uk ) .
the precursor mass tolerance was set at 7 
 ppm , and fragment ion mass tolerance set at 0.5 amu .
the only permitted 
 post - translational modifications were n - terminal acetylation and cysteine 
 carbamidomethylation , while a maximum of two miscleavages were allowed . 

quantification of the predefined targeted peptides was carried out 
 using sieve version 2.0 ( thermo fisher scientific ) using two seed 
 files , one for each dme region of interest , containing retention time 
 and m / z information .
the precursor 
 mass tolerance was set to 5 ppm , and the minimal intensity for alignment 
 was set at 100  000 , with intensities derived from the first 
 monoisotopic peak .
data from sieve were exported to excel 2010 ( microsoft , 
 redmond , wa ) for calculation of light to heavy peptide ratios . for 
 protein quantification ,
light to heavy protein ratios were calculated 
 within samples by summing average intensity values for all light peptides 
 for each protein , then dividing by the corresponding heavy value . 

light to heavy protein ratios for technical replicates were averaged 
 then biological replicates normalized to the average of control , before 
 calculation of fold changes . for calculation of statistical significance
, 
 normalized values were log2-transformed and then analyzed by unpaired 
 student s t test ( fdr = 0.5% ) using prism 
 6 ( graphpad , la jolla , ca ) ; * p < 0.05 , * * p < 0.01 , * * * p < 0.001 .
a workflow 
 schematic of the thr / sim in vivo silac 
 approach is shown in figure 1a .
liver lysates 
 were combined 1:1 with a liver lysate from metabolically labeled in vivo silac animals , which served as an internal standard 
 for each experimental sample .
gel electrophoresis was selected for 
 sample preparation , as it allowed for fractionation and enrichment 
 of dmes at the protein level .
this selection was based on a pilot 
 comparison of the sds - page in - gel digestion method to fasp . in the 
 former ,
dmes were resolved by sds - page into two defined molecular 
 weight regions which were excised for analysis ; dme region 1 ( 6640 
 kda ) was expected to contain adh , aldh , cyp , fmo , ugt , and ephx enzymes , 
 while dme region 2 ( 4022 kda ) was expected to contain akr , 
 gst , n - acetyltransferase ( nat ) , and sulfotransferase ( sult ) enzymes , 
 based on calculated mw values ( figure 1b ) . 

protein in excised bands was reduced , alkylated , and digested with 
 trypsin according to published protocols .
a total of 64 dmes were identified by peaks 
 analysis after the two - sample in - gel procedure , compared to 49 following 
 the single - sample fasp procedure ( supporting information tables s-1s-3 ) .
( a ) metabolically 
 labeled mouse liver tissue lysates were combined 1:1 with experimental 
 sample lysates for sds - page .
excised bands were processed by in - gel 
 trypsin digestion for ls  ms / ms analysis .
heavy / light ratios 
 from the first monoisotopic peaks were calculated for each peptide 
 of interest in individual samples , with fold changes across samples 
 calculated using the ratio / ratio value .
( b ) the gel bands excised , 
 region 1 ( 6640 kda ) and region 2 ( 4022 kda ) , are predicted 
 to contain the dmes of interest .
similar to the principles of stable isotope dilution lc  ms 
 methods , the accuracy of measurements in thr / sim are highly dependent 
 on the reliable detection of heavy stable isotope signals .
it was 
 therefore important to characterize the liver proteome of the spike - in 
 reference lysate to determine which dmes could be monitored .
as detailed 
 above , sds - page and in - gel digestion of an in vivo silac liver sample permitted identification of a total of 64 dmes 
 ( 2 adh , 7 akr , 11 aldh , 23 cyp , 1 ephx , 2 fmo , 9 gst , 0 nat , 2 sult , 
 and 7 ugt ) by unique peptides ( fdr = 0.1% ) .
in addition , due to the 
 high degree of identity shared by certain enzymes , some were unidentifiable 
 by unique peptides but were nonetheless retained as protein groups , 
 for example , cyp2a4/2a5 .
peptides without lysine were removed from 
 the list , as were those with 10log p values 
 < 17.3 ( fdr = 0.1% ) .
heavy - only  peptide list 
 was converted to a list of silac peptide pairs by addition of the 
 predicted counterpart light members .
the utility of these peptide 
 pairs for quantification was then stress - tested as follows .
an unlabeled 
 liver lysate was mixed with the in vivo silac liver 
 lysate at ratios of 1:1 , 1:4 , and 1:16 in duplicate for processing 
 and analysis by lc 
the ms data were analyzed in sieve 
 using the silac peptide pair list as a seed file ( retention time window 
 of 5 min ) .
the average ratio value from technical replicates 
 was then log4 transformed to evenly distribute the three data points , 
 allowing calculation of an equally weighted r value . only those peptides showing strong linearity ( r > 0.9 ) were retained .
the data output from sieve , along 
 with 
 calculations of r values , can be found 
 in supporting information tables s-4 and 
 s-5 .
approximately 57% of identified peptides survived this filtration 
 step ( supporting information figure s-1 ) . 

seed files containing m / z and retention 
 time windows for each peptide can be found in supporting information tables s-6 ( dme region 1 ) and s-7 ( dme 
 region 2 ) .
an example of thr / sim in vivo silac 
 is shown in figure 2 .
three cyp1a2 peptides 
 were used to define cyp1a2 levels , one of which eluted at 37.9 min 
 and another at 94.7 min ( figure 2a ) .
although 
 some interfering signals were observed , these could be separated from 
 the signals of interest by retention time gating and the matching 
 of internal standard signals . for one of these peptides ,
nsiqditsalfk , 
 an r value for linearity of 0.998 was 
 observed ( figure 2b ) and the identities of 
 both the light and heavy forms could be confirmed by their ms2 ( figure 2c and d ) .
( a ) using a 
 5 min retention time window ( gray boxes ) , nontarget signals 
 could be effectively gated from nsiqditsalfk and fltnnnsaidk 
 peptide pair quantification .
( b ) dilution linearity of the nsiqditsalfk 
 peptide pair achieved r of 0.998 , while 
 ms2 spectra were acquired for both ( c ) light and ( d ) heavy forms , 
 confirming their identity . to compare the output of thr / sim in vivo silac to 
 western blotting , we assessed the cyp induction profile in mice treated 
 with a potent and specific inducer of the constitutive androstane 
 receptor , tcpobop .
animals ( n = 3 ) were administered 
 the compound in corn oil , and , after 7 days , livers were harvested 
 for analysis .
western blotting for cyp indicated strong induction 
 of multiple family members , compared to vehicle control ( figure 3a ) .
depending on the origin of antisera , as well 
 as results of previous studies ( not shown ) , the exact identity of 
 individual enzymes may or may not be known . with our in - house panel 
 of antibodies , up - regulation
could be demonstrated for cyp1a1 ( 46.5-fold ) , 
 cyp1a2 ( 5.1-fold ) , cyp2b10 ( 362.9-fold ) , and 3a11 ( 5.7-fold ) , while 
 down - regulation could be demonstrated for cyp2e1 ( 0.8-fold ) , as these 
 superfamily members are predictably and reproducibly identified . for 
 other antisera , such as cyp2c and cyp2d ,
the precise identities of 
 the proteins detected are unknown , so , although inductions are observed , 
 these are not attributable to any family member in particular . for 
 t - hr
/ sim - in vivo silac , data for individual animals 
 were acquired and processed separately , and then average fold changes 
  sd calculated at the last stage , to account for biological 
 variability .
cyp1a2 ( 3.5-fold ) , cyp2a4/5 ( 16.2-fold ) , cyp2c29 ( 15.7-fold ) , 
 cyp2c37 ( 9.5-fold ) , cyp2d22 ( 2.5-fold ) , cyp3a13 ( 5.3-fold ) , and cyp8b1 
 ( 3.1-fold ) were significantly up - regulated ( figure 3b ) .
although not statistically significant ( p = 0.08 ) , cyp2e1 levels were decreased ( 0.87-fold ) . due to the low 
 abundance of cyp1a1 , cyp2b10 , and cyp3a11 in the spike - in standard , 
 these enzymes could not be quantified by lc 
nevertheless , 
 there was close agreement between methods of changes in cyp1a2 and 
 cyp2e1 expression .
moreover , western blotting suggested induction 
 of unconfirmed cyp2c , cyp2d , and cyp3a family members , while lc 
ms / ms 
 demonstrated induction of cyp2c29 , cyp2c37 , cyp2d22 , and cyp3a13 .
cyp isoform in tcpobop - treated mice ( n = 3 ) , as compared 
 to corn oil vehicle - treated control animals ( n = 
 3 ) , were measured by ( a ) western blot and ( b ) thr / sim - in vivo silac ( black bars , corn oil ; gray bars , tcpobop ) . following western 
 blot of pooled samples ,
chemilluminescent signal was used to quantify 
 fold changes in particular bands ( red boxes ) , with values presented 
 alongside .
note 
 that , in the control , some protein levels were below the limit of 
 detection by western blotting ( e.g. , cyp2b10 ) and therefore the fold 
 changes calculated may not be accurate . for
thr / sim - in vivo silac , the number of unique peptides used to calculate fold change 
 is given ( gray text ) , and as samples for individual animals were analyzed 
 separately , error bars are a reflection of biological variability , 
 as well as technical error . to provide additional confidence that the assay was reliable , 

we 
 extracted data for four proteins / protein families which are not expected 
 to change significantly following tcpobop dosing : pro / albumin , calreticulin , 
 -tubulin , and -tubulin .
of the 
 other quantifiable phase i enzymes , aldh1a1 ( 3.1-fold ) and fmo5 ( 3.1-fold ) 
 were significantly up - regulated , while aldh2 ( 0.4-fold ) and aldh6a1 
 ( 0.5-fold ) were significantly down - regulated ( figure 4a ) . for phase ii , gsta3 ( 1.6-fold ) , gstm1 ( 7.5-fold ) , gstt3 
 ( 7.0-fold ) , sult3a1
( 4.7-fold ) , ugt1a1/2 ( 2.9-fold ) , ugt2b34 ( 3.8-fold ) 
 and ugt2b36 ( 1.8-fold ) were significantly up - regulated ( figure 4b ) .
ugt2a3 ( 0.8-fold ) was the only enzyme for which 
 significant down - regulation occurred .
thr / sim - in vivo silac 
 analysis of modulation of 
 additional dmes by tcpobop .
( a ) other phase i and ( b ) phase ii enzymes 
 were quantified .
through the combination 
 of in vivo silac and label - free 
 approaches we have created a streamlined and simple targeted proteomics 
 workflow permitting the quantitative analysis of 51 dmes in the mouse . 

this technique has utility in the investigation of pharmacodynamics , 
 particularly when the potential for , or occurrence of , drug 
upstream , the use of metabolically labeled 
 isotopic tissue as an internal standard reduces experimental variation 
 derived from sample processing and ionization .
this is especially 
 important when error - prone methods such as in - gel digestion are used , 
 and when the difference between experimental groups is relatively 
 small .
downstream , the strategy resembles that of stable isotope dilution 
 lc - ms for quantification of small molecules .
the narrow retention 
 time and m / z windows reduce the 
 influence of potential contaminant signals during data processing , 
 while data analysis is simplified in comparison to more conventional 
 silac procedures as only a predetermined list of proteins / peptides 
 of interest is measured , permitting immediate and intuitive interpretation .
one limitation of the thr / sim in vivo silac approach 
 is that changes in expression can not be monitored in the dme for which 
 heavy isotope labeled ms2 are not acquired .
a potential solution to 
 this problem would be to rerun a database search on the experimental 
 ms files and , where ms2 for light peptides from nontargeted dme can 
 be detected , manually interrogate the primary raw file , referencing 
 the light signal to other heavy peptides eluted within the same time 
 frame , or to total ion current as typically performed with label - free 
 proteomics .
caution must be reserved 
 that such quantification may be subject to sources of peptide bias , 
 such as differential recovery during sample processing and differential 
 efficiency during ionization .
although in the current study we believe 
 high resolution sim mode is sufficient to monitor the levels of 51 
 dme proteins , mainly due to their higher relative abundance , the method 
 can be easily adapted to an mrm mode where the precursor and fragment 
 ion transitions are monitored .
we have applied the thr / sim - in vivo silac workflow 
 to the analysis of dme changes in response to the car activator , tcpobop . 
 with an ec50 of approximately 100 nm , maximally effective 
 dose of 3 mg / kg , and response duration of greater than 20 weeks in 
 the mouse ,
it acts as a mitogen , nongenotoxic tumor - promoter and complete carcinogen . in the liver , in studies variously employing tcpobop and/or car 
 knockout mice as tools to characterize the car - dependent gene battery , 
 up - regulation of transcription of mrna has been demonstrated for cyp1a1 , 
 1a2 , 2a4 , 2a5 , 2b9 , 2b10 , 2b13 , 2c29 , 2c37 , 2c55 , 2c65 , 2f2 , and 3a11 , 
 with decreases seen for 4a10 and 4a31 . at the protein level
, widespread cyp induction has 
 been demonstrated by western blot and 
 in a postdigest o labeling study , where cyp1a2 , 2a4/5 , 
 2b10 , 2b20 , 2c29 , 2c37 , 2c38 , 3a11 , and 39a1 were shown to be up - regulated , 
 with cyp2c40 , 2e1 , 3a41 , and 27a1 down - regulated . in the current study , we detected significant up - regulation 
 of cyp1a2 , 2a4/2a5 , 2c29 , 2c37 , 2d22 , 3a13 , and 8b1 and nonsignificant 
 down - regulation of cyp2e1 , broadly in agreement with the literature , 
 although we did not see the previously reported changes in cyp2f2 . 

this could be due to post - transcriptional or post - translational regulation , 
 or variation in genetic background .
our data conflict with the report 
 of cyp27a1 down - regulation , as we detected 
 a nonsignificant increase .
we did not observe any significant changes 
 in adh or akr but , to our knowledge , the only previously reported 
 car / tcpobop target within these superfamilies is akr1b7 , which we did not detect and were therefore unable 
 to quantify .
the only other reported car / tcpobop - regulated members 
 of the phase i metabolism superfamilies under study are aldh1a1 , aldh1a7 , 
 and fmo5 , which are up - regulated at the mrna level .
our data agree with those for aldh1a1 and fmo5 , although 
 we could not detect aldh1a7 protein .
in previous reports , aldh1b1 , 
 2 , 6a1 , and 7a1 have shown a car / tcpobop - dependent down - regulation .
our data agree completely with these observations , although only 
 the more pronounced decreases in aldh2 and 6a1 achieve significance . for phase ii , at the mrna level , car
/ tcpobop can upregulate gsta1 , 
 a2 , a4 , m1 , m2 , m3 , m4 , t1 and t3 , sult1a1 , 1e1 , 2a1 , 2a2 , 3a1 and 
 5a1 , ugt1a1 , 1a9 , 2b34 , 2b35 and 2b36 , and down - regulate ugt2a3 and 
 3a1/2 . in agreement with the literature , we observed significant 
 induction of gsta3 , m1 , t3 and sult3a1 , and nonsignificant induction 
 of gstm2 and t1 , and sult1a1 . for ugt , we detected induction of ugt1a1/2 , 
 2b34 , and 2b36 , with repression of 2a3 , all in agreement with previous 
 ( mrna ) reports .
our findings agree with the only report we are aware of regarding 
 phase ii protein induction by car / tcpobop , that of ugt1a1 .
therefore , as the vast majority of car targets 
 have been established as such from mrna studies , it is noteworthy 
 that , for many of these targets , we have provided the first evidence 
 for their modulation at the protein level . moreover , despite the influence 
 of post - transcriptional and post - translational regulation , we have 
 demonstrated that the car activation signature is essentially conserved 
 from mrna to protein . while we found that the thr / sim approach 
 is satisfactory for the 
 analysis of 51 dme proteins using a ltq - orbitrap , other recently developed 
 ms2 based targeted
quantitative proteomics approaches such as swath , ms , and parallel reaction monitoring ( prm ) may provide an alternative if a quadrupole - orbitrap or quadrupole - time - of - flight 
 mass spectrometer is available .
in particular , the prm approach has 
 recently been shown to provide better sensitivity and specificity 
 than sim .
precursor ion pairs that are preisolated by a quadrupole , accumulated 
 and fragmented in a c - trap .
it is therefore anticipated that the number 
 of dme proteins measured could be potentially improved using the prm 
 approach . in conclusion ,
the application of thr / sim - in vivo silac for quantification of 51 dme presented here 
 constitutes a 
 comprehensive means of profiling altered capacity for drug metabolism 
 in the mouse .
an appreciation of this capacity is valuable when carrying 
 out pharmacokinetic studies in this model organism , when up- or down - regulation 
 of dme expression due to drug treatment or genetic manipulation has 
 a bearing on results .
moreover , this approach could provide an additional 
 measure of the ddi potential of a candidate therapeutic compound during 
 preclinical development .
as modulation of dmes typically requires 
 upstream activation of one or more nhr by direct binding to chemical 
 agents , species differences in drug / receptor affinity mean that caution 
 must be reserved in drawing conclusions from studies in model organisms . 

conceivably , thr / sim - in vivo silac could be used 
 to measure ddi potential in nhr - humanized mice , such as the car / pxr 
 double humanized line , constituting a 
 more - accurately modeled in vivo approach and thereby 
 reducing the gap between preclinical and clinical evaluation of ddi .
 the modulation of 
 drug metabolism enzyme ( dme ) expression by therapeutic 
 agents is a central mechanism of drug  drug interaction and 
 should be assessed as early as possible in preclinical drug development . 
 
 direct measurement of dme levels is typically achieved by western 
 blotting , qpcr , or microarray , but these techniques have their limitations ; 
 antibody cross - reactivity among highly homologous subfamilies creates 
 ambiguity , while discordance between mrna and protein expression undermines 
 observations . 
 the aim of this study was to design a simple targeted 
 workflow by combining in vivo silac and label - free 
 proteomics approaches for quantification of dmes in mouse liver , facilitating 
 a rapid and comprehensive evaluation of metabolic potential at the 
 protein level . 
 a total of 197 peptides , representing 51 phase i and 
 phase ii dmes , were quantified by lc - ms / ms using targeted high resolution 
 single ion monitoring ( thr / sim ) with a defined mass - to - charge and 
 retention time window for each peptide . in a constitutive androstane 
 receptor ( car ) activated mouse model , comparison of thr / sim - in vivo silac with western blotting for analysis of the 
 expression of cytochromes p450 was favorable , with agreement in fold - change 
 values between methods . 
 the thr / sim - in vivo silac 
 approach therefore permits the robust analysis of multiple dme in 
 a single protein sample , with clear utility for the assessment of 
 the drug  
 drug interaction potential of candidate therapeutic 
 compounds . 
Introduction
Materials and Methods
Results
Discussion