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Targeted Quantitative Proteomics for the Analysis of 14 UGT1As and -2Bs in Human Liver using nanoUPLC-MS/MS with Selected Reaction Monitoring John K. Fallon, Hendrik Neubert, Ruth Hyland, Theunis C Goosen, and Philip C. Smith J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr4004213 • Publication Date (Web): 26 Aug 2013 Downloaded from http://pubs.acs.org on September 2, 2013

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Journal of Proteome Research

Targeted Quantitative Proteomics for the Analysis of 14 UGT1As and 2Bs in Human Liver using nanoUPLC-MS/MS with Selected Reaction Monitoring

John K. Fallon,† Hendrik Neubert,‡ Ruth Hyland,‡ Theunis C. Goosen,‡ and Philip C. Smith*,†

†

Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 ‡

Dept. of Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., Cambridge, MA and Groton, CT.

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KEYWORDS UGT1As, UGT2Bs, targeted quantitative proteomics, SIL, nanoUPLC-MS/MS, microsomes, S9s

ABSTRACT Targeted quantitative proteomics using heavy isotope dilution techniques is increasingly being used to quantify proteins, including UGT enzymes, in biological matrices. Here we present a multiplexed method using nanoLCMS/MS and multiple reaction monitoring (MRM) to quantify fourteen UGT1As and UGT2Bs in liver matrices. Where feasible, we employ two or more proteotypic peptides per protein, with only four proteins quantified with only one proteotypic peptide. We apply the method to analysis of a library of 60 human liver microsome (HLM) and matching S9 samples. Ten of the UGT isoforms could be detected in liver and the expression of each was consistent with mRNA expression reported in the literature. UGT2B17 was unusual in that ~30 % of liver microsomes had no or little (97 % purity, concentration ±25 %). A library of 60 commercially prepared individual human liver microsome samples and their matching S9 fractions (n=59; the S9 of one sample was unavailable) (BD Gentest™, BD Biosciences) was supplied by Pfizer.

Stable isotope labeled internal standards Some SIL peptide internal standard solutions, received from the manufacturer (Thermo, AQUA QuantPro Peptides) in the earlier part of the studies, had been dissolved in 5 % acetonitrile and to these was added additional acetonitrile (30 % or 50 % of the total volume) to ensure the solubilization of hydrophobic peptides. 5


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Solutions received later in the studies contained 20 % acetonitrile and were used without modification. Thermo supplies SIL standards as 1 nmoles/vial with calibration based on amino acid analysis. The working solution was prepared by combining equal amounts of peptides (n=30) (see Table 1 for complete list) to achieve concentrations of 0.1 pmol/µL which allowed the addition of 1 pmol (10 µL) of the proteotypic peptides to be added to each sample. The working solution was prepared fresh each day of use. Selection of proteotypic peptides (assessed via BLAST search) was done as described previously23,24 with consideration of potential missed tryptic cleavage sites and M content which were avoided. Although peptides less than 15 AA acids in length are preferred and having at least two peptides per protein is preferred, this was not always feasible with this comprehensive method for an entire protein family such as the UGT1As and UGT2Bs. When performing in silico predictions prior to ordering and evaluating a longer list of crude potential proteotypic peptides, estimates of relative hydrophobicity (SSRCalc, Manitoba Centre for Proteomics and Systems Biology) and electrospray efficiency (ESPPredictor, GenePattern: Proteomics, Broad Institute, MIT) were useful to avoid peptides that were poorly retained on reversed phase LC or too hydrophobic, or had poor electrospray efficiency, respectively.

Sample preparation and digestion Liver samples were stored at -80 °C until thawed for analysis. Total protein concentrations of all samples (n=60 individual microsomal, n=59 individual S9 and n=2 mixtures of individual microsomal) were checked using the BCA protein assay kit and, unless ≥ 50 % ≤ of the nominal concentration (the nominal concentration of all samples was 20 mg/mL), values obtained were used to adjust UGT isoform concentrations determined. Where total protein concentrations were ≥ 50 % ≤ of the nominal value adjustments were made in the initial dilution of the samples. For sample preparation microsomal samples were diluted to ~ 1 mg/mL (1 µg/µL) with 50 mM ammonium bicarbonate and S9 samples were diluted to ~ 5 mg/mL (5 µg/µL). Tryptic extension peptides (x 3; 1 pmol of each, representing UGTs 1A1, 1A9 and 2B7) (with tryptic peptide double heavy isotope 6


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labeled) and β-casein (0.5 µg) were added to all samples as indicators of successful digestion (see Table 2 for tryptic extension and β-casein peptide sequences; two peptides were used for β-casein). The tryptic extension peptides were added first (to Fisher PCR tubes, 0.2 mL; see below) and evaporated to dryness, to remove acetonitrile, in a ThermoSavant ISS110 SpeedVac before proceeding as described below. The β-casein was added in 50 mM ammonium bicarbonate solution before denaturation and reduction. The single SIL peptides, for quantification, were added after digestion. The usual number of samples per batch was sixty four, including two HLM samples that contained high and low concentrations of UGT1A1 as QC samples prepared in triplicate. Aliquots of diluted sample (for microsomal samples 20 µg nominal total protein was used, for S9 samples 100 µg was used; see above for dilutions) were added to the PCR tubes (0.2 mL) containing 50 µL of 50 mM ammonium bicarbonate. These small tubes were used so that a 96-well Isotemp Thermal Mixer plate could be employed for batches. 10 µL of 40 mM DTT and 10 µL β-casein (0.05 µg/µL) were then added. Samples were denatured and reduced for 40 min at 60 °C. After cooling to room temperature, 10 µL of 135 mM IAA was added and samples were incubated for 30 min in the dark at room temperature. Trypsin was then added (10 µL; 0.1 µg/µL in 50 mM acetic acid) to give a trypsin:nominal protein ratio of 1:20 (w/w) for microsomal and recombinant samples and 1:100 (w/w) for S9 samples (extra trypsin was added to some additional S9 samples [2 samples x 3 replicates] to give a 1:20 (w/w) ratio and found to give similar results to when the higher ratio was used). Samples were then vortexed and digested at 37 °C for 4 h using the IsoTemp Thermal Mixer (Fisher Scientific, Pittsburg, PA) mixing at 300 rpm. The reaction was stopped by the addition of 75 µL of ice cooled acetonitrile to each sample. A 10 µL aliquot of a mixture of SIL peptides standards (1 pmol of each, working solution was prepared fresh with each batch; see above) was then added to each sample. The contents of each vial were transferred to 0.6 mL LoBind Epp tubes and the vials were rinsed with a further 1 x 75 µL ACN. Samples were evaporated to dryness in a ThermoSavant ISS110 SpeedVac and reconstituted in 50 µL of water/acetonitrile/formic acid 98/2/0.1 (i.e. modified mobile phase A; see below). Samples were then vortexed at 600 rpm for 10-15 min in the 7


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IsoTemp Thermal Mixer. The samples were centrifuged for 5 min at 12000 rpm and supernatants were transferred to vials containing silanized inserts for nanoUPLC-MS/MS analysis.

MRM Selection The two best MRMs for each peptide were chosen by an iterative process with the help of Skyline 1.1 software (University of Washington, WA). Initial collision energies (CEs) of a number of MRMs (e.g. 20 MRMs) were first predicted by the software. Optimization of CEs was enabled by importing data from the Analyst 1.5 software into Skyline at each stage of the process. The two best MRMs for each β-casein peptide (Table 2) were chosen in a similar way using a trypsin digested standard.

Instrumentation and UPLC-MS/MS conditions Analysis was performed on a nanoACQUITY binary pump system (Waters, Milford, MA) coupled to an AB SCIEX QTRAP 5500 hybrid mass spectrometer equipped with a NanoSpray III source. An uncoated PicoTip® emitter (20 µm inner diameter, 10 µm tip; New Objective, Woburn, MA) was used to produce the nanospray. Control was by Analyst 1.5 software (AB SCIEX, Framingham, MA) and nanoACQUITY UPLC Console. Mobile phase A consisted of 1 % acetonitrile and 0.1 % formic acid. Mobile phase B was 100 % acetonitrile. An injection volume of 2 µL was loaded onto a Symmetry C18 trap column, 180 µm x 20 mm, 5 µm particle size (Waters) at a trapping flow of 15 µL/min of mobile phase A for 1 min. Peptides were eluted from the trap column and separated at a flow rate of 2 µL/min on a BEH130 C18 column, 150 µm x 100 mm, 1.7 µm particle size (Waters). For seven of nine batches of samples and quality controls (see Assay Qualification) prepared the separation conditions were 100 % A at start, to 58 % A at 24 min, 5 % A at 24.5 min for 3 min and 100 % A at 28 min for 7 min (total run time was 35 min). For the other two batches the separation conditions were 100 % A at start, to 65 % A at 20 min, 5 % A at 20.5 min for 3 min and 100 % A at 24 min for 7 min (total run time 31 min). The analytical column temperature was set to 35 °C.

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MS analysis was conducted in the positive mode with ion spray voltage at 4000. GS1 (nitrogen), after optimization, was set at 15 (arbitrary units) and the interface heater temperature was 150 °C. CUR, EP and CXP were 20, 10 and 10, respectively. Target scan time was 1.5 sec with a scheduled MRM detection window of 60 sec. Pause time between MRMs was 3 ms. The nanospray, as mentioned above under Instrumentation, was produced with an uncoated PicoTip® emitter (20 µm inner diameter, 10 µm tip). Declustering potential (DP) values recommended by Skyline 1.1 were used in the analyses, as alteration of this produced little change. MRM data processing was by MultiQuant 2.0 (AB SCIEX). Peaks were smoothed prior to integration and area ratios of unlabeled/SIL peptides were determined using the sum of the MRMs monitored.

Assay Qualification Optimization of denaturation temperature and digestion time Denaturation temperature was examined by analyzing pooled HLMs in triplicate at two temperatures, 60 and 95 °C. Optimum digestion time was determined by comparing pooled HLMs digested for 0 h, 5 min, 1, 2, 4, 8, and 24 h. Samples were prepared in triplicate for each time point, except for the 5 min samples which were prepared in duplicate.

Selectivity, sensitivity and linearity Interferences from solvents, matrices and SIL peptides were investigated by preparation and monitoring of blank samples over the course of the experiments. SIL peptide standards were spiked into RLMs (10 µg) in decreasing amounts to give duplicate samples representing concentrations from 100 to 0.15 pmol/mg protein. The limit of detection (LOD) was determined as the lowest concentration giving a signal to noise ratio of three.

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A similar experiment using recombinant UGT samples was undertaken. For this 12 rUGTs, namely rUGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17, were combined into a solution and diluted into RLMs to give samples containing a range of UGT concentrations. The total protein amount (rUGT + RLM) in each sample was 10 µg. An assumption of each initial recombinant sample containing 10 % of the UGT of interest was made (the samples were reported to contain 5 to 15 % of the protein of interest; personal communication, BD Biosciences). The concentration range prepared thus represented samples of 138, 55.2, 27.6, 13.8, 5.52, 2.76, 1.1, 0.552, 0.276 and 0.11 pmol/mg protein. All samples, except the highest concentration sample, were prepared in duplicate. The experiment served to give an estimate of the UGT content of the recombinant samples (SIL peptide standards were also added) and an overview of linearity and sensitivity.

Intra- and interday variability Intraday precision was determined by analyzing five replicates of each of the two QC HLM samples described in Materials (representing high and low concentration UGT1A1). 20 µg of total protein was used for each replicate (the total protein content of each sample had been determined using the BCA protein assay kit). For interday variability the two QCs were analyzed in triplicate with each batch of samples prepared (n=9). They were injected at the beginning and at the end of each batch, thus also providing information on the stability of prepared samples in the autosampler at 12 °C.

Digestion reproducibility, injection reproducibility and stability of prepared samples Digestion reproducibility was studied in the quality controls by 1) examining combined MRM area ratios of one β-casein peptide to the other, 2) examining MRM area ratios of double SIL peptides (i.e. from tryptic extension peptides) to corresponding single SIL peptides and 3) examining area ratios of β-casein MRMs to combined MRM areas of the single SIL UGT2B7 peptide IEIYPTSLTK (chosen because of good peak signal intensity). 10

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Injection reproducibility studies included injecting one of each of the two QC samples (10 µg total HLM protein was used in each of these samples) at 7 h intervals five times. This also provided data on sample stability over approximately 28 h in the autosampler at 12 °C.

Selection of Peptide Data for Reporting UGT Protein Concentrations Following the calculation of individual peptide concentrations, peptides were chosen for the reporting of protein concentrations. Rationale for selection of peptides included stability to digestion (relative to other peptides), LOQ and statistical analysis of the variance distribution for each peptide using the interday quality control data set (i.e. triplicate samples of the two QCs prepared with nine batches; n=9).

RESULTS A representative total ion chromatogram for one of the HLM samples is shown in Figure 1. Peaks representing SIL peptides are dominant as these were present in excess (at a concentration of 1 pmol per 20 µg total protein sample; 50 pmol/mg) when compared to those of endogenous peptides. The elution time range of peptides is from approximately 10 to 25 min. Of the 30 peptides examined, 26 were considered suitable for use in the analyses (Table 1). Figure 2 shows extracted ion chromatograms from examples of the representative UGT1A1 high and low HLM QCs (HLMs 74 and 81, respectively).

Assay Qualification Optimization of denaturation temperature and digestion time Of the two denaturation temperatures tested 60 °C was found to give greater concentrations for all isoforms (e.g. 10-20 % higher) and to give higher peak responses (data not shown). Following digestion of HLMs for various times, 4 h was chosen as the optimum digestion time. Digestion time profiles for individual peptides are shown in Figure 3. For UGTs 1A1 and 1A4 one of the three peptides for 11

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each of these isoforms was found to give lower concentrations at longer digestion times (Figure 3), indicating probable degradation. These peptides were thus not used for reporting protein concentrations for their respective isoforms.

Selectivity, sensitivity and linearity SIL standards, tryptic extension peptides, β-casein and buffers and reagents used in sample preparation were found not to interfere with chromatographic and mass spectral selectivity. The LODs for all SIL peptides, except peptides 8 (UGT1A7) and 25 (UGT1A8), in RLMs were found to be

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