1

Introduction

Glucuronidation is a very important pathway for the detoxification and elimination of many endogenous and exogenous compounds and is catalysed by the UDP-glucuronosyltransferases (UGTs), which are membrane-bound enzymes (Tukey & Strassburg 2000; Wells et al. 2004). There are four UGT families and members of the UGT1A family share the same last four exons with the other three families (exons 2-5) with only the first exon differentiating between the four families. Members of the UGT1A1 family have been shown to be involved in a

number of important physiological and pharmacological processes including conjugation of bilirubin, detoxifica-tion of potential carcinogens, conjugation of estradiol, and other critical phase II metabolic steps (Senafi et al. 1994; Bosma et al. 1995; Malfatti et al. 2005).

Detection and quantification of the protein profiles in cells, tissues or body fluids provides an accurate methodology for detecting changes in cellular dynamics in health and disease. Due to the high amount of sequence identity among the various members of the UGT1A family, quantitation using antibodies is

ReseaRch aRTIcLe

Quantitation of UGT1A1 in human liver microsomes using stable isotope-labelled peptides and mass spectrometry based proteomic approaches

Chitra Sridar1, Imad Hanna2, and Paul F. Hollenberg1

1Department of Pharmacology, University of Michigan, Ann Arbor, MI, USA and 2Novartis Institutes for Biomedical Research, East Hanover, NJ, USA

abstract1. UDP-glucuronosyltransferases (UGTs) are a group of drug-metabolizing enzymes that catalyse the conjugation

of endogeonous compounds and xenobiotics to yield hydrophilic glucuronides which subsequently undergo excretion. This report describes an approach for the identification and accurate quantitation of human UGT1A1 in complex biological matrices using liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) analysis of protein digests.

2. A stable isotope-labelled (SIL) peptide of a unique peptide spanning residues 54–69 in exon 1 of the human UGT1A1 protein with the sequence RIYLSADPALVVIEHG was synthesized. The peptide sequence synthesized was in the reverse order of the human peptide with the stable isotope-labels in the amino acid arginine (13C

615N

4) resulting

in an increase in the mass of the SIL peptide of 10 amu, from 1753 to 1763. The SIL peptide was quantitated by injecting increasing concentrations of the peptide into the LC-MS to obtain a standard curve.

3. The labelled peptide along with precursor ion monitoring was used to quantify the levels of UGT1A1 in commercial recombinant preparations (supersomes) and individual human liver microsomal samples and pooled human liver micrsomes obtained from BD Biosciences.

4. Glucuronidation activity studies were performed, which demonstrated a positive correlation between enzyme activity levels and the UGT1A1 content in the liver microsomes obtained from individual human donors.

Keywords: acetonitrile, dithiothreitol, extracted ion chromatogram, iodoacetamide, human liver microsomes, stable isotope labelled, single ion monitoring, single reaction monitoring, trifluoroacetic acid, uridine diphosphoglucuronic acid, UDP-glucuronosyltransferase

Address for Correspondence: Dr. Paul F. Hollenberg, Department of Pharmacology, The University of Michigan, 1150 West Medical Center Dr, Ann Arbor, MI 48109-5632, USA. Tel.: 734 764 8166, Fax: 734 763 5387 E-mail: phollen@umich.edu

(Received 13 June 2012; revised 31 July 2012; accepted 03 August 2012)

Xenobiotica, 2012; Early Online: 1–10© 2012 Informa UK, Ltd.ISSN 0049-8254 print/ISSN 1366-5928 onlineDOI: 10.3109/00498254.2012.719089

Xenobiotica

00

00

1

10

13June2012

31July2012

03August2012

0049-8254

1366-5928

© 2012 Informa UK, Ltd.

10.3109/00498254.2012.719089

2012

Quantitation of UGT1A1 using stable isotope labelling

C. Sridar et al.

Xen

obio

tica

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Mic

higa

n U

nive

rsity

on

12/0

4/12

For

pers

onal

use

onl

y.

2 C. Sridar et al.

Xenobiotica

challenging due to cross reactions among the UGT1A enzymes. Also, despite great advances in protein technology over the past decade, membrane-bound proteins still represent one of the most difficult classes of proteins to analyse quantitatively. This is in part due to their limited solubility and the refractory nature of many membrane proteins to proteolytic digestion. To date, although several different approaches have been tried in attempts to solve these problems, none have proven to be totally satisfactory. Two-dimensional gel electrophoresis in combination with tandem MS has been one of the approaches attempted to be used (Shevchenko et al. 1996; Unlü et al. 1997). However, this approach is labour-intensive, difficult to reproduce, and is relatively difficult to automate, especially with the membrane proteins. In addition, quantitation by mass spectrometry has proven to be a relatively challenging analytical problem because of the potential for suppression of ionization by the co-eluting proteins during the electrospray process. To overcome this analytical problem, peptides labelled with stable isotopes in combination with mass spectrometry have become a widespread technology and appear to offer a promising method for the absolute quantitation of proteins (Barr et al. 1996; Kuhn et al. 2004; Kamiie et al. 2008). A report by Kamii et al. (2008) shows the use of stable isotope-labelled (SIL) peptides to quantitate 34 different transporter proteins in the livers, kidneys and brains of mice. Stable isotope-labelled peptide analogues can also be used as internal standards in quantitative studies of proteins because their chemical and physical properties are similar to those of the naturally occurring peptides with the only difference being in the mass. The quantitation of proteins and peptides by mass spectrometry is dependent on the use of a standard curve which can be obtained by using a stable isotope-labelled peptide. This peptide is then used as the internal standard by spiking the analytical sample with a known amount of the SIL peptide. The ratio of the endogenous peptide to the synthetic peptide is then determined by MS analysis and the absolute amount of the peptide derived from the protein of interest can be calculated. The approach of using selected ion monitoring to quantify proteins and phosphoproteins has previously been used by Gerber et al. (2003). Selected ion monitoring is performed by restricting the acquisition mass range around the m/z value of the ion(s) of interest (Lange et al. 2008). The potential of single reaction monitoring (SRM)-based proteomics to map out the whole yeast proteome to detect and quantify any protein expressed at a concentration above single-digit copies/cell has been demonstrated by Picotti et al (2009). Thus, quantitation using the isotope dilution approach holds great promise in the quantitation of diagnostic or prognostic protein markers and in investigating cellular mechanisms of drug actions.

A signature peptide is a peptide that is unique to the protein of interest that is obtained by the proteolytic or chemical digestion of the protein (Zhang et al. 2004).

Thus, in this study, quantitation of native or recombinant UGT1A1 protein in human liver microsomal samples and in insect-cell membrane preparations, respectively, using a signature peptide was developed using liquid chromatography. This approach relied on tandem mass spectrometric measurements of samples that have been spiked with a SIL peptide that is the reverse of the sig-nature peptide for UGT1A1 as an internal standard. The ratio of the areas of the precursor ions derived from the endogenous peptide and the SIL peptide provide a method to measure the amount of the protein of interest in the various samples.

Materials and methods

MaterialsAcetonitrile (ACN), dithiothreitol (DTT), trifluoroace-tic acid (TFA), iodoacetamide (IAA), urea, ammonium bicarbonate, β-estradiol, β-estradiol-3(β-d-glucuronide) sodium salt, UDP-glucuronic acid (UDPGA) and alam-ethicin were purchased from Sigma-Aldrich (St Louis, MO, USA). Sequencing grade modified trypsin was purchased from Promega (Madison, WI, USA). Insect cell mem-brane preparations containing recombinant UGT1A1 (Supersomes) and pooled human liver microsomes (HLM) were purchased from BD Biosciences (Woburn, MA, USA). The HLM obtained from BD Biosciences are a pooled sample prepared from frozen human tissues. The HLM from individual donors used for the assays of β-estradiol glucuronidation were from our lab and had been prepared previously by Dr. John Teiber (Teiber & Hollenberg 2000).

Identification and selection of a signature peptide for UGT1A1UGT1A1 supersomes (20 µg) were dissolved in 100 mM ammonium bicarbonate buffer and reduced by adding 5 mM (final concentration) DTT. The protein was then alkylated by the addition of 10 mM IAA and incubated for 30 min at 37°C. The protein samples were then digested using sequencing grade modified trypsin (1:20 trypsin: protein ratio) in 100 mM aqueous ammonium bicarbon-ate (pH 8.0). The samples were incubated overnight at 37°C. After digestion, 1 µL of 10% TFA was added to the samples and they were centrifuged at 13,200×g for 10 min in an Eppendorf centrifuge. The supernatants were transferred into new vials and analysed using a LC/ESI-MS-MS ion trap mass spectrometer as described below. Separation of the peptides was performed on a C18 column (Phenomenex, Jupiter 5 µ). The HPLC mobile phases consisted of 100% water containing 0.25% formic acid/0.25% TFA (solvent A) and 100% ACN containing 0.25% formic acid/0.25% TFA (solvent B). Gradient elu-tion of the column was performed as follows: the pro-portion of solvent B was increased linearly from 10 to 30% over 50 min and then linearly to 95% over 90 min. It was maintained at 95% solvent B for 10 min and then re-equilibrated at 90% solvent A for 10 min. The flow rate was 0.3 mL/min.

Xen

obio

tica

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Mic

higa

n U

nive

rsity

on

12/0

4/12

For

pers

onal

use

onl

y.

Quantitation of UGT1A1 using stable isotope labelling 3

© 2012 Informa UK, Ltd.

The electrospray MS was operated in the positive mode. Tandem mass spectrometric analysis was performed on a LCQ Deca ion trap mass spectrometer with an activation time of 30 ms, activation Q of 0.3, normalized collision set at 35% and dynamic exclusion width set at 1.5. The mass spectrometer was operated in the data dependent mode in which the initial MS scan recorded the mass to charge (m/z) ratio of the ions over the mass range 150–2000 kDa and then the six most abundant ions were automatically selected for subsequent collision-induced dissociation. The MS/MS spectra were compared with a database downloaded from the National Center of Biotechnology Information (NCBI) using TurboSEQUEST software (ThermoFinnigan, Waltham, MA, USA). The peptides were identified by correlating their fragmentation spectra with the amino acid sequences obtained from the NCBI protein database. Several selection criteria were used to select the signature peptide. Peptides with multiple proline residues were not considered. The peptide selected did not contain any residues such as cysteine or methionine, since they have a tendency to undergo oxidation. Also, peptides shorter than five amino acids were excluded.

Synthesis of a SIL peptide for use in calibrationThe SIL peptide was synthesized and purified at the University of Michigan Protein Structure Facility using standard techniques. The SIL peptide was synthesized in the reverse order of the signature peptide with the labelling done in the amino acid arginine. Synthesis in the reverse order of the peptide was chosen because the core-facility had the stable-labelled arginine avail-able and having the label at the front end of the peptide is more stable than at the back end. Peptide purity was >94% as determined by the protein core facility based on HPLC analysis of the peptide. Stock solutions of the peptide were prepared in 50% acetonitrile/water. All further dilutions of the stock solutions were made with deionized water. The sequence of the isotope-labelled peptide was RIYLSADPALVVIEHG where the amino acid R contained six 13C and four N15 atoms, thus leading to an increase in the mass of the peptide of 10 amu (1753 to 1763). The eluant from the HPLC was introduced into the source of the MS via the electrospray interface. For the construction of standard curves using the SIL peptide, eight different dilutions of the working standard solution were prepared from the stock solutions and injected into the LC-MS/MS and MS/MS spectra were acquired. The peptide eluted at 22.8 min and the mass of the peptide [M+H]+ exhibited a m/z of 1762.9 and the precursor ion of the SIL peptide [M+2H]+2 had a m/z of 882. Dilutions of the working standard solutions were prepared from the stock solutions using deionized water and stored at −20°C. The final concentrations of the internal standards plotted against the peak areas gave a linear response and were used to construct the standard curves. The mass of the SIL peptide did not change during incubation with trypsin indicating that the arginine at the N-terminus of the SIL peptide did not get clipped.

Tryptic digestion of the UGT1A1 supersomesIn order to quantify the amount of UGT1A1 in the super-somes, aliquots containing 20 µg of the supersomes were spiked with 5 µL of 68 pmol/µL (a total of 340 pmol) of the SIL peptide and digested as indicated above. The samples were concentrated to a volume of 60 µL using an Amicon centrifugal filter (Millipore Corporation, Bellerica, MA, USA) with a molecular weight cutoff of 3000. A 50 µL ali-quot was then injected onto the HPLC-MS/LCQ-DecaXP. The precursor ion selected for analysis of the UGT1A1 supersomes was the [M+2H]+2 ion of the signature pep-tide at m/z 877.09 and [M+2H]+2 for the corresponding stable isotope-labelled peptide at m/z 882.08. Both native and labelled peptide formed abundant [M+2H]2+ ions at m/z 877 and 882, respectively. The production of these ions (m/z 877→1753, 882→1763) was monitored for both peptides and provided the basis for the quantitation (Seibert et al. 2009). To calibrate the SIL peptide in the presence of UGT1A1 supersomes, 20 µL of supersomes were spiked with SIL peptide with concentrations rang-ing from 10 fmol to 800 fmol. Digestion and LC-MS analy-sis of the samples were performed as described above.

Tryptic digestion of the HLMTotal protein concentrations in the HLM from individual donors were determined using the BCA protein assay (Pierce Chemical). The concentration of the protein in the three individual donor samples used was 30, 42 and 59 mg/mL. The concentration of the protein in the HLM obtained from BD Biosciences was 20 mg/mL. A 10 µL aliquot of the HLM was diluted with 100 mM ammonium bicarbonate buffer (pH 8.0) to a volume of 200 µL. The samples were then treated with 6M urea and reduced with 5 mM DTT for 30 min at 65°C. The reduced and denatured protein samples were alkylated with IAA at a final concentration of 10 mM for 20 min at 37°C in the dark. Following alkylation, the mixtures were diluted with 50 mM ammonium bicarbonate (pH 8.0) to a final volume of 7 mL and then concentrated through an Amicon centrifugal filter (Millipore Corporation) with a 3000 molecular weight cutoff membrane to decrease the urea concentration. The volumes were reduced to 300 µL and the samples were then spiked with 364 pmol of the internal standard. Digestion of the alkylated microsomal proteins was carried out by adding trypsin at a 100:1 microsomal protein/enzyme ratio. The samples were incubated overnight in a shaking water bath at 37°C. The digestions were terminated by the addition of 1 µL of 10%TFA. The samples were vortexed and centrifuged at 13000×g in an Eppendorf centrifuge for 10 min, and the supernatants were used for LC-MS/MS analysis. LC-MS analysis was carried out using a Shimadzu LC system equipped with Shimadzu’s SCL-10A system controller and a SIL-10AD auto injector. The tryptic peptides were separated on Phenomenex Jupiter 5 µ C18 (150 × 2.00 mm) column. The peptides from the trypsin-digested UGT1A1 supersomes and the HLM were then subjected to LC-MS analysis as described. The mobile phases and the flow

Xen

obio

tica

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Mic

higa

n U

nive

rsity

on

12/0

4/12

For

pers

onal

use

onl

y.

4 C. Sridar et al.

Xenobiotica

rate were as described above. The peptides were sepa-rated using a linear gradient of 10–50% B over a period of 90 min. This shallow gradient provides better chromato-graphic separation.

Data analysisThe peptides eluting from the LC from both the UGT1A1 supersomes and the HLM were analysed on an LCQ Deca ion trap mass spectrometer equipped with an electro-spray ion source (ThermoFinnigan). ESI ionization was performed in the positive ion mode and the mass spec-trometer was operated in the data dependent mode in which first an initial MS scan recorded the mass to charge (m/z) ratio of ions over the mass range 150–2000 kDa, and then the six most abundant ions were automatically selected for subsequent collision-induced dissociation. Precursor activation was performed with an activation time of 30 ms, activation Q of 0.3 and the normalized col-lision was set at 35%. The dynamic exclusion width was set at 1.5. The flow rate was 0.3 mL/min and the gradi-ent is as described above. Data acquisition and analysis were performed using both Xcalibur software (v. 1.2, ThermoQuest, Manchester, UK) and SEQUEST Bioworks (Thermo Electron Corp). The proteins and peptides were identified automatically by the SEQUEST BioWorks computer program, which correlated the experimental tandem mass spectra against theoretical tandem mass spectra from amino acid sequences obtained from the National Center for Biotechnology Information (NCBI) sequence database. Also, manual inspection of the peptide identifications from the MS/MS spectrum was performed in order to ensure that the major MS/MS frag-mentation peaks matched the theoretical peaks.

Interday and intraday assay variationThe reproducibility of the assays was validated using intra- and interday calibration curves. To examine intraday variability, calibration curves for two experi-ments done in triplicate using pooled HLM from human liver micrsomes (samples 115, 127 and 136) and BD Biosciences were prepared and analysed on the same day. Interday variability experiments were carried out in a similar manner with standard curves being prepared and analysed on three different days using digests of pooled HLM from BD Biosciences.

β-Estradiol glucuronidation assayUGT1A1 catalyses the glucuronidation of estradiol resulting in the formation of estradiol glucuronide. Experiments were conducted to confirm that the rate of glucuronidation was proportional to the concentra-tion of the UGT1A1 protein which was determined using ESI-LC/MS. The incubations contained UGT 1A1 supersomes (0.05 mg/mL) mixed with 50 mM potassium phosphate buffer (pH 7.1), 1 mM MgCl

2 and alamethicin

(25 µg/mL) and placed on ice for 30 min. For determina-tion of kinetic parameters, increasing concentrations of β-estradiol (concentrations ranging from 5 to 50 µM) in

0.5 µL of DMSO were added to a final incubation volume of 100 µL. The samples were preincubated in a 37°C water bath and the reactions were initiated by the addition of UDPGA to a final concentration of 5 mM. The reactions were terminated after 30 min by the addition of an equal volume of ice-cold acetonitrile. The incubation samples were spun at 13,200×g for 10 min in a centrifuge and the supernatant was transferred to a fresh tube and evapo-rated to dryness using a stream of nitrogen. The samples were then reconstituted in 90/10 % (v/v) acetonitrile/water and analysed by ESI-LC/MS using a Phenomenox C18 Luna 3 µ phenyl-hexyl (150 × 2.00 mm) column. The mobile phase consisted of 5 mM ammonium acetate (pH 4.5), 0.1 % acetic acid (A) and acetonitrile/methanol (90/10, v/v), 0.1% acetic acid (B). The column was eluted isocratically with 5% B for 10 min and the concentration of B was increased linearly to 90% B over 17 min and then held at 90% B for 10 min before returning to initial conditions. The mass spectrometer was operated in the negative mode with acquisition by single ion monitoring (SIM) of m/z 447 and a product ion at m/z 271.

β-Estradiol glucuronidation assays using HLM were also performed to validate the quantitation results obtained above. The total protein content of the indi-vidual HLM was first determined using the BCA assay (Smith et al. 1985). The protein concentrations were determined to be 30, 42 and 59 mg/mL for HLM-115, HLM-127 and HLM-136, respectively. The protein con-tent of the pooled HLM obtained from BD was 20 mg/mL. To quantify the UGT1A1 contents in the micro-some samples, 10 uL aliquots of the samples were used. Furthermore, to quantitate the UGT1A1 enzyme activity in the HLM by glucuronidation of β-estradiol, each of the microsomal samples was incubated with increasing concentrations of β-estradiol (0.5–100 µM). The samples were preincubated in a 37°C water bath and the reac-tions were initiated by the addition of UDPGA to a final concentration of 5 mM. The reactions were terminated and estradiol 3-glucuronidation activity was measured as described above. The kinetic parameters, K

m and V

max

were determined by nonlinear regression analysis using GraphPad Prism version 5.0 for Windows (GraphPad Software Inc., San Diego, CA, USA). The data were fitted to both the Michaelis–Menton and Hill Equations and the best fit was to the Hill Equation.

Results and discussion

The use of SIL peptides has recently been widely employed for the quantitation of proteins in biological samples. Quantitation can be performed by analysing the parent and the product ion mass spectra of the analyte and an internal standard. Quantitation can be done by comparing the precursor ion monitoring of the native to the labelled peptide which gives a specific fragmentation pattern and by comparing the peak height or peak area of the labelled to the unlabelled peptide. The UGT1A1 enzyme is involved in catalysing the glucuronidation

Xen

obio

tica

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Mic

higa

n U

nive

rsity

on

12/0

4/12

For

pers

onal

use

onl

y.

Quantitation of UGT1A1 using stable isotope labelling 5

© 2012 Informa UK, Ltd.

of bilirubin. It is also involved in glucuronidation of 2-hydroxyestrogenic catechols, anthraquinones, cou-marins, flavonoids and phenolic compounds. Drugs that inhibit UGT1A1 could result in hyperbilirubinemia and may reduce the hepatic clearance of coadministered drugs that are cleared by UGT1A1. An accurate measure-ment of UGT1A1 expression and other UGTs in the liver by proteomic approaches is necessary in order to better predict the contribution of these enzymes to the disposi-tion of new drugs and to gauge the effect of the inhibition of one, or all of these enzymes on the overall clearance. This also allows for the estimation of the contribution of UGT1A1 to hepatic clearance by scaling of in vitro parameters obtained from recombinant preparations. This is analogous to what is currently done for CYPs since their absolute levels (nmol/mg microsomal protein) in HLM can be measured.

Identification of a signature peptide for UGT1A1The UGT1A1 supersomes obtained from BD Biosciences were digested using the protocol described in the Methods. Supersomes (20 µg) were digested with trypsin and about 10 µg of digested protein was injected into the electrospray LC-MS. Sequest Bioworks 3.2 software was used to determine the peptide fragments and the Sequest search identified UGT1A1 with sequence cover-age of 70%. The peptides obtained from the digest were submitted to BLAST analysis using the human Swiss-Prot database to identify a unique peptide which could be used to positively identify the UGT1A1 protein and could also be used as an internal standard for quantita-tion. The peptide, Gly-His-Glu-Ile-Val-Val-Leu-Ala-Pro-Asp-Ala-Ser-Leu-Tyr-Ile-Arg, spanning residues 54–69 from exon 1 of UGT1A1with a m/z of 1752.8 seemed to be unique and no similar tryptic peptides were found when a search was performed against the proteins in the NCBI database. The peptide also lacked the following post-translational modifications as was determined using the software indicated in parentheses: N-linked glycosylation (NetNGlyc), O-linked glycosylation (NetOGlyc), meth-ylation (MeMO), acetylation (NetAcet), palmitoylation (NBA-Palm), myristoylation (NMT), phosphorylation (Kinase Phos or Net Phos) and sumoylation (SUMOsp). These various software programs predict the posttransla-tional modifications within given proteins and a warning is displayed if a signal peptide is not detected. Manual validation of the spectrum was done to verify that the SEQUEST-assigned sequence was consistent with the MS/MS spectrum for that peptide. Figure 1 shows the MS/MS spectrum of the signature peptide. The mass of the singly charged peptide ([M+H]+) is m/z 1752.8. The spectrum shows the product ion for the doubly-charged protonated molecule of the signature peptide at m/z 877.1. The intense peaks at m/z 1695.65, 1429.66, 1217.48, 1118.48, 1005.31, 934.4, 819.48, 748.27, 635.3, 564.350 and 288.329 are found for the signature peptide obtained at the time of the eluting peak confirming the peptide sequence.

The above unique peptide was synthesized and quan-titated using electrospray LC-MS to obtain a standard curve by using increasing concentrations of the peptide. However, on analysis, the ion count did not increase in a linear fashion with the increase in peptide concentration (data not shown). This is possibly because of interference in the ionization that contributes to the ion suppression effect. Hence, a stable isotope-labelled peptide was syn-thesized for the same peptide. High labelling efficiency is a key for successful quantitation and thus labelling of arginine with 13C

6 and 15N

4 was used and the peptide

was synthesized in the reverse order. The reverse order for the synthesis of the peptide was chosen because the core-facility had the arginine-labelled amino acid avail-able for peptide synthesis. The peptide internal standard was then analysed by MS/MS to examine the peptide fragmentation patterns.

To optimize the parameters for the LCQ Deca mass spectrometer for the quantitation of UGT1A1, an ali-quot of the SIL peptide was infused directly into the ESI source via a syringe. MS and MS/MS spectra were obtained and suitable fragment ions were chosen for quantitation. Figure 2 shows the MS/MS spectrum of the internal standard. The SIL peptide did exhibit a slightly longer retention time (30 s) when compared with the signature peptide seen in the UGT1A1 digest (data not shown). This is possibly because the SIL peptide has a different sequence since it is in the reverse order of the peptide of interest. However, we did ensure that the retention time scale was aligned for all experiments that were performed. The precursor ion of the SIL peptide was monitored on the basis of the generation of the ion m/z 1762.79→882.08. As seen in Figure 2, many of the b and y-ions could be identified and exhibited good intensi-ties. All of the b-ions showed an increase in 10 amu as expected because of the stable isotope-labelling in argi-nine. In addition, a few of the the a-ions (molecular mass of the N-terminal amino acid-CHO) were also observed.

Tryptic digestion of UGT1A1 supersomesThe UGT1A1-containing supersomes were digested with trypsin and spiked with the SIL internal standard to quantitate the amount of UGT1A1 protein in the mixture. In complex mixtures such as these, co-eluting peptides present a very challenging problem. These are peptides that have distinctly different molecular weights but have similar retention times. Use of the data-dependent acquisition mode, where the precursor ion of an acquired scan is selected regardless of the presence of any other co-eluting peptides, helps greatly in peptide identifi-cation and quantitation. Both the native and labelled peptides formed abundant [M+2H]2+ ions at m/z 877 and 882, respectively. The production of these ions (m/z 1753→877, 1763→882) was monitored for both peptides. In Figure 3, the top chromatogram shows the extracted ion chromatogram (EIC) at m/z of 887 of the native pep-tide corresponding to [M+2H]+2. The bottom chromato-gram shows the EIC at m/z of 882 of the internal standard

Xen

obio

tica

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Mic

higa

n U

nive

rsity

on

12/0

4/12

For

pers

onal

use

onl

y.

6 C. Sridar et al.

Xenobiotica

peptide corresponding to [M+2H]+2. The ratio of the areas under the curves for the peptide peaks of the digested protein (upper chromatogram) and the SIL peptide (lower chromatogram) gives a value of 0.43 which allows us to calculate from our standard curve a concentration of UGT1A1 in the supersomes of 11.2 pmol/15µg or 747 pmol/mg.

To calibrate the technique using the SIL peptide, vari-ous standard solutions were added to the same amount of supersomes and analysed by LC-ESI-MS/MS as described above. As shown in Figure 4, the amount of the SIL peptide (in pmol) added in the supersome samples is shown on the X-axis and the Y-axis shows the peak

area for the protein digest. The SIL peptide gave a linear response within the tested range of 10–800 fmol. In this range, the correlation coefficient was r2 = 0.98. The stabil-ity of the SIL peptide was verified by adding the SIL pep-tide to the recombinant UGT1A1 preparation samples before and after adding trypsin for the digestion. No sig-nificant change was observed. The results show that the peak area to concentration ratios for the SIL peptide is consistent with the linear response curve.

Tryptic digestion of HLMIn order to quantitate the UGT1A1 protein in a sample of HLM, the SIL peptide was added to the tryptic digest of

Figure 1. The MS/MS spectrum of the signature peptide of UGT1A1 after tryptic digestion and analysis by LC-MS/MS. UGT1A1 supersomes were digested with trypsin and the LC-MS/MS analysis was performed as described in Materials and Methods. The figure shows the singly charged fragment ions from the peptide with m/z 1752.8 and a doubly charged protonated molecule with m/z 877.1. Several of the y and b-ions identified are shown in the red and blue colors, respectively.

Figure 2. The MS/MS spectrum of the SIL peptide. Conditions for the dilution of the synthetic peptide and its injection and analysis by LC-MS/MS are described in Materials and Methods. The figure shows the MS/MS spectrum for the singly charged fragment ions from the SIL peptide (13C615N4 RIYLSADPALVVIEHG) with the m/z 1762.79 and a doubly charged protonated molecule with m/z 882.1. Several of the y and b-ions identified are shown in the red and blue colors, respectively. The a-ion is derived from the b-ion by the loss of CHO.

Xen

obio

tica

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Mic

higa

n U

nive

rsity

on

12/0

4/12

For

pers

onal

use

onl

y.

Quantitation of UGT1A1 using stable isotope labelling 7

© 2012 Informa UK, Ltd.

the microsomes and processed for analysis as described previously. SEQUEST analysis of the MS/MS data for the HLM identified about 2200 proteins with 0.5 restrictions on the protein and peptide probability settings. After filtering out low-confidence proteins and peptides, we obtained about 800 proteins. Manual validations of the spectra were done to ascertain whether the SEQUEST-assigned sequences were consistent with the MS/MS

spectra for the peptides. All of the abundant peaks were validated to either the b or y-ions. Proteins were then identified by matching the MS/MS spectra from the digest to the theoretical spectra from the NCBI database. Sequence coverage for the identified proteins was found to vary between 10 and 60%. This variation could be due to a number of factors including: the accessibility of the protein to trypsin, peptide fragmentation and the drying of the sample. The variability in the sample preparation was investigated by running the same sample three times and comparing the results. The average variability ranged from 4 to 12% indicating that the sample preparation pro-cedure involving reduction, alkylation and digestion and drying was relatively consistent and reproducible.

Figure 5A shows the total-ion chromatogram for the full scan MS of the HLM digest. Figures 5B and 5C are the spectra for the precursor ions at m/z 876.8 and 882.0, respectively, eluting off the column on which we had injected the HLM digest spiked with the SIL peptide. The ratio of the areas gives a value of 0.214 (data not shown). Based on the peak areas of the precursor ions of the endogenous peptide and the labelled peptide and the amount of protein injected, a concentration of 110 pmol of UGT1A1/mg of protein was calculated. This is compa-rable to the amount of P450 2E1 seen in the microsomal protein from human liver as reported by Seibert et al (2009) where a concentration of 127 pmol of P4502E1/mg of mircrosomal protein was calculated for the human liver.

Intraday and interday assay variation Intra- and interday variation, which is represented by %CV was calculated for all the four HLM. The intraday variation data for quantification of UGT1A1 %CV for the four HLM samples were calculated from three replicates done two times. The vaues were 1.7, 7.7, 4.0 and 21.1%CV for HLM-115, HLM-127, HLM-136 and HLM-BD, respec-tively. The interday variation data for the same four HLM samples done on 3 different days were 7.1, 8.0, 4.0 and 15.1%CV.

β-Estradiol glucuronidation assayGlucuronidation catalysed by UGTs is a major clearance pathway for many endogenous and xenobiotic com-pounds in humans. The UGT1A1 enzyme contributes to the glucuronidation of endogenous bilirubin as well as drugs such as estradiol, irinotecan and buprenorphine (Liston et al. 2001). Thus, a decrease or increase in the lev-els or activity of UGT1A1 due to genetic polymorphisms, drug-drug interactions or disease conditions may lead to the buildup of endogenous substances and/or drugs to toxic levels. Estradiol is glucuronidated at the 3-posi-tion by UGT1A1 (Senafi et al. 1994) and at the 17-position by several UGTs (Gall et al. 1999). In order to investigate the relationship between glucuronidation activity and the concentration of UGT1A1 protein as measured by LC-MS/MS, the estradiol-3-glucuronidation assay was performed on UGT1A1 supersomes and the pooled

Figure 3. Tryptic digestion and LC-MS/MS analysis of UGT1A1-containing supersomes in the presence of the SIL peptide. A 20 µg sample of UGT1A1 supersomes was digested as described in Materials and Methods. The sample was spiked with 340 pmol of the SIL peptide and injected onto the LC-MS for quantitation. The top panel shows the ion chromatogram of m/z 1753→877 corresponding to the [M+2H]+2 for the native peptide. The bottom panel shows the ion chromatogram of m/z 1763→882 corresponding to the [M+2H]+2 for the labeled peptide.

Figure 4. Calibration curve for the SIL peptide in the presence of UGT1A1 supersomes. 20 µg of supersomes was digested as described in Materials and Methods. The digested UGT1A1 supersomes were then spiked with varying concentrations of the SIL peptide (10–800 fmol). Dilution of the SIL peptides was carried out as described in the Materials and Methods. The samples were then injected onto the LC-MS for quantitation. Analysis of the samples was carried out as described in Materials and Methods. The X-axis shows the amount of the peptide injected. The Y-axis gives the peak area for that sample. Each data point is the average of three measurements with the error bars representing the standard deviation.

Xen

obio

tica

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Mic

higa

n U

nive

rsity

on

12/0

4/12

For

pers

onal

use

onl

y.

8 C. Sridar et al.

Xenobiotica

human liver sample obtained from the BD Biosciences as well as three additional human liver samples from inde-pendent donors and compared with the protein concen-trations as measured by LC-MS/MS. The standard curve for β-estradiol glucuronidation was then shown to be lin-ear over the range of 5–1000 pmol (data not shown). The linearity of product formation with respect to incubation time was also established. The metabolite production

with β-estradiol is approximately linear for 60 min. The correlation coefficients for both assays were r2 = 0.90 and 0.91 (data not shown), respectively.

Using the conditions described above, enzyme kinetic parameters were determined for the recombinant UGT1A1-containing supersomes and HLM from BD, and for three additional human liver microsomal preparations.

Figure 5. LC-MS/MS data from a tryptic digestion of human liver microsomes. The human liver microsomes were spiked with the SIL peptide and the digestion was carried out as described in Materials and Methods. Panel A shows the total ion chromatogram of the human liver microsomes. Panel B shows the MS spectrum for the precursor ion at m/z 876.8 for the peptide with m/z 1753 and panel C shows the MS spectrum for the precursor ion at m/z 882.0 for the SIL peptide with m/z 1763.

Table 1. Determination of UGT1A1 concentrations for human liver microsome samples and the kinetic parameters for the β-glucuronidation activity of the same microsomal samples.

Samples

Concentration of UGT1A1 in

pmol/mg protein

Estradiol β-glucuronidation

Km

in µM

Vmax

pmol/min/mg

protein

UGT 1A1 supersomes

747 ± 102.2 0.45 ± 0.04 1079 ± 411.5

HLM-115 8.6 ± 0.15 18.1 ± 7.3 20.45 ± 4.6HLM-127 19.5 ± 1.5 25.7 ± 0.4.2 50.35 ± 7.89HLM-136 52.5 ± 2.1 45.2 ± 30.47 111.4 ± 22.4HLM-BD 110 ± 22.9 0.777 ± 0.07 210.6 ± 6.41The mass spectrometeric method for determining UGT1A1 concentrations for the supersomes and human liver microsome samples is described in Materials and Methods. The values represent the average and standard deviation of two independent experiments done in triplicates. UGT1A1 and human liver microsome samples were incubated with β-estradiol to determine the kinetic parameters as described in Materials and Methods. Each data point for the β-estradiol glucuronidation activity assay represents the average and standard deviation of two experiments done in duplicate.

Figure 6. Correlation between the concentrations of UGT1A1 in the human liver microsomal samples as measured by LC-MS/MS and the Vmax values estimated from glucuronidation of estradiol. Kinetic constants for estradiol glucuronide formation by the human liver microsomal samples were determined as described in the Materials and Methods and are shown in Table 1, as are the UGT1A1 concentrations. The data from Table 1 were then plotted as shown here. The data represent the means and standard deviations for measurements performed twice in duplicate.

Xen

obio

tica

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Mic

higa

n U

nive

rsity

on

12/0

4/12

For

pers

onal

use

onl

y.

Quantitation of UGT1A1 using stable isotope labelling 9

© 2012 Informa UK, Ltd.

The data were fitted to the Michaelis–Menton equa-tion and the best fit was to the Hill Equation. Using the labelled peptide and the procedure described above, the quantitation of UGT1A1 by LC-MS/MS was carried out for the four different HLM samples. As shown in Table 1, the concentration of UGT1A1 in the human liver samples when measured by the LC-MS/MS method varied from 8.6 to 110 pmol/mg of protein between the four samples, thus, there was a 12-fold difference in the concentrations. Since glucuronidation at the 3-β position has been shown to be primarily catalysed by UGT1A1, estradiol was used as a probe to determine the enzyme activity levels in the human liver samples. The HLM in our studies exhibited K

m values ranging from 0.77 to 45 µM and the V

max values

ranged from 20 to 210 pmol/min/mg proteins. Previous studies by Mano et al (2005) and Soars et al. (2003) have shown the K

m values range from approximately 16.8 to 30

µM, respectively. The large range in Km

values in our study could be because the samples are from different human samples and the condition of the patient (diseased or oth-erwise) from which these samples were obtained is not known. A plot of the concentration of UGT 1A1 in the HLM as measured by our mass spectral approach versus the V

max values for β-glucuronidation (Figure 6) demonstrates

a direct correlation between the two with an r2 of 0.99.Quantitation of protein expression under normal as

well as diseased, induced or stressed conditions helps in predicting the variability of individuals with respect to their responses to drugs. The quantitation of specific cellular proteins is important for studying cellular functions and predicting responses to drugs and other xenobiotics. Some of the challenging issues during quantitation include: co-eluting peptides, nonspecific cleavage, posttranslational modification, single-nucleotide polymorphisms and several others. However, stable isotope labelling and tan-dem mass spectrometry have been used since 1987 and are being used increasingly in proteomic studies (Yergey et al. 1987; Kuhn et al. 2004; Lane et al. 2007). Similar stud-ies using heavy labelled peptides for the absolute quanti-fication of UGTs 1A1 and 1A6 have previously been done by Fallon et al (2008). The method involved stable isotope labelled synthetic peptide with multiple reaction monitor-ing using LC-MS/MS analysis. Harbourt et al have have quantified different isoforms of UGT1A1 using nanobore high-performance liquid chromatography coupled to a linear ion trap time-of-flight mass spectrometer (Harbourt et al. 2012). The results presented here differ from those of Ohtsuki et al. (2012) and Schaefer et al. (2012) in that we demonstrate a quantitation technique that can be per-formed on a much less sophisticated and less expensive instrument that is more readily available in more labora-tories. In addition, we also demonstrate validation of the quantitation results using the β-glucuronidation assay. Although our study analysed only one UGT isoform in a single run using SIM, it should be possible to quantify multiple UGT or CYP isoforms using this approach.

In summary, the amount of UGT1A1 was measured by stable isotope dilution mass spectrometry using a

unique tryptic peptide from the protein and selected ion monitoring analysis. The peak area for the protein digest was normalized to the peak area of the internal standard. It is important to note that the method presumes there is complete tryptic digestion of the proteins of interest and on performing the quantitation within a concentration range which exhibits a linear response. Tryptic digestion of complex mixtures such as supersomes and HLM and subsequent analysis using mass spectrometry having a somewhat limited detection range presents significant challenges with respect to determining how much inter-nal standard to add to the sample. The amount may vary significantly for different proteins. Thus, selected ion monitoring helps in detecting the intact peptide mass and one of the specific fragment ions for the peptide pro-vides additional specificity and selectivity to the peptide detection during the experiment. The results presented here indicate that the method described above provides good accuracy and a linear calibration curve and that this approach may be applied for the quantitation of proteins in complex mixtures such as plasma samples and tissue homogenates. Thus, quantitation using isotope-labelled peptides is expected to have excellent application to the pharmaceutical sciences and will allow for more precise prediction of in vivo drug–drug interactions.

acknowledgements

This work was supported in part by National Institutes of Health Grant CA 16954 and Novartis.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

ReferencesBarr JR, Maggio VL, Patterson DG Jr, Cooper GR, Henderson LO, Turner

WE, Smith SJ, Hannon WH, Needham LL, Sampson EJ. (1996). Isotope dilution–mass spectrometric quantification of specific proteins: model application with apolipoprotein A-I. Clin Chem 42:1676–1682.

Bosma PJ, Chowdhury JR, Bakker C, Gantla S, de Boer A, Oostra BA, Lindhout D, Tytgat GN, Jansen PL, Oude Elferink RP. (1995). The genetic basis of the reduced expression of bilirubin UDP-glucuronosyltransferase 1 in Gilbert’s syndrome. N Engl J Med 333:1171–1175.

Fallon JK, Harbourt DE, Maleki SH, Kessler FK, Ritter JK, Smith PC. (2008). Absolute quantification of human uridine-diphosphate glucuronosyl transferase (UGT) enzyme isoforms 1A1 and 1A6 by tandem LC-MS. Drug Metab Lett 2:210–222.

Gall WE, Zawada G, Mojarrabi B, Tephly TR, Green MD, Coffman BL, Mackenzie PI, Radominska-Pandya A. (1999). Differential glucuronidation of bile acids, androgens and estrogens by human UGT1A3 and 2B7. J Steroid Biochem Mol Biol 70:101–108.

Gerber SA, Rush J, Stemman O, Kirschner MW, Gygi SP. (2003). Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc Natl Acad Sci USA 100:6940–6945.

Harbourt DE, Fallon JK, Ito S, Baba T, Ritter JK, Glish GL, Smith PC. (2012). Quantification of human uridine-diphosphate glucuronosyl transferase 1A isoforms in liver, intestine, and

Xen

obio

tica

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Mic

higa

n U

nive

rsity

on

12/0

4/12

For

pers

onal

use

onl

y.

10 C. Sridar et al.

Xenobiotica

kidney using nanobore liquid chromatography-tandem mass spectrometry. Anal Chem 84:98–105.

Kamiie J, Ohtsuki S, Iwase R, Ohmine K, Katsukura Y, Yanai K, Sekine Y, Uchida Y, Ito S, Terasaki T. (2008). Quantitative atlas of membrane transporter proteins: development and application of a highly sensitive simultaneous LC/MS/MS method combined with novel in-silico peptide selection criteria. Pharm Res 25:1469–1483.

Kuhn E, Wu J, Karl J, Liao H, Zolg W, Guild B. (2004). Quantification of C-reactive protein in the serum of patients with rheumatoid arthritis using multiple reaction monitoring mass spectrometry and 13C-labeled peptide standards. Proteomics 4:1175–1186.

Lane CS, Wang Y, Betts R, Griffiths WJ, Patterson LH. (2007). Comparative cytochrome P450 proteomics in the livers of immunodeficient mice using 18O stable isotope labeling. Mol Cell Proteomics 6:953–962.

Lange V, Picotti P, Domon B, Aebersold R. (2008). Selected reaction monitoring for quantitative proteomics: a tutorial. Mol Syst Biol 4:222.

Liston HL, Markowitz JS, DeVane CL. (2001). Drug glucuronidation in clinical psychopharmacology. J Clin Psychopharmacol 21:500–515.

Malfatti MA, Ubick EA, Felton JS. (2005). The impact of glucuronidation on the bioactivation and DNA adduction of the cooked-food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in vivo. Carcinogenesis 26:2019–2028.

Mano Y, Usui T, Kamimura H. (2005). In vitro inhibitory effects of non-steroidal antiinflammatory drugs on UDP-glucuronosyltransferase 1A1-catalysed estradiol 3beta-glucuronidation in human liver microsomes. Biopharm Drug Dispos 26:35–39.

Ohtsuki S, Schaefer O, Kawakami H, Inoue T, Liehner S, Saito A, Ishiguro N, Kishimoto W, Ludwig-Schwellinger E, Ebner T, Terasaki T. (2012). Simultaneous absolute protein quantification of transporters, cytochromes P450, and UDP-glucuronosyltransferases as a novel approach for the characterization of individual human liver: comparison with mRNA levels and activities. Drug Metab Dispos 40:83–92.

Picotti P, Bodenmiller B, Mueller LN, Domon B, Aebersold R. (2009). Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics. Cell 138:795–806.

Schaefer O, Ohtsuki S, Kawakami H, Inoue T, Liehner S, Saito A, Sakamoto A, Ishiguro N, Matsumaru T, Terasaki T, Ebner T. (2012). Absolute quantification and differential expression of drug transporters, cytochrome P450 enzymes, and UDP-glucuronosyltransferases in cultured primary human hepatocytes. Drug Metab Dispos 40:93–103.

Seibert C, Davidson BR, Fuller BJ, Patterson LH, Griffiths WJ, Wang Y. (2009). Multiple-approaches to the identification and quantification of cytochromes P450 in human liver tissue by mass spectrometry. J Proteome Res 8:1672–1681.

Senafi SB, Clarke DJ, Burchell B. (1994). Investigation of the substrate specificity of a cloned expressed human bilirubin UDP-glucuronosyltransferase: UDP-sugar specificity and involvement in steroid and xenobiotic glucuronidation. Biochem J 303(Pt 1):233–240.

Shevchenko A, Jensen ON, Podtelejnikov AV, Sagliocco F, Wilm M, Vorm O, Mortensen P, Shevchenko A, Boucherie H, Mann M. (1996). Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. Proc Natl Acad Sci USA 93:14440–14445.

Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. (1985). Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85.

Soars MG, Ring BJ, Wrighton SA. (2003). The effect of incubation conditions on the enzyme kinetics of udp-glucuronosyltransferases. Drug Metab Dispos 31:762–767.

Teiber JF, Hollenberg PF. (2000). Identification of the human liver microsomal cytochrome P450s involved in the metabolism of N-nitrosodi-n-propylamine. Carcinogenesis 21:1559–1566.

Tukey RH, Strassburg CP. (2000). Human UDP-glucuronosyltransferases: metabolism, expression, and disease. Annu Rev Pharmacol Toxicol 40:581–616.

Unlü M, Morgan ME, Minden JS. (1997). Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18:2071–2077.

Wells PG, Mackenzie PI, Chowdhury JR, Guillemette C, Gregory PA, Ishii Y, Hansen AJ, Kessler FK, Kim PM, Chowdhury NR, Ritter JK. (2004). Glucuronidation and the UDP-glucuronosyltransferases in health and disease. Drug Metab Dispos 32:281–290.

Yergey AL, Esteban NV, Liberato DJ. (1987). Metabolic kinetics and quantitative analysis by isotope dilution thermospray LC/MS. Biomed Environ Mass Spectrom 14:623–625.

Zhang F, Bartels MJ, Brodeur JC, Woodburn KB. (2004). Quantitative measurement of fathead minnow vitellogenin by liquid chromatography combined with tandem mass spectrometry using a signature peptide of vitellogenin. Environ Toxicol Chem 23:1408–1415.

Xen

obio

tica

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Mic

higa

n U

nive

rsity

on

12/0

4/12

For

pers

onal

use

onl

y.