Variability in mass spectrometry-based quantification of clinically relevant drug transporters and drug metabolizing enzymes

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1 Christine Wegler Supporting information 1 (13) Variability in mass spectrometry-based quantification of clinically relevant drug transporters and drug metabolizing enzymes Christine Wegler 1,2, Fabienne Z. Gaugaz 1, Tommy B. Andersson 2, Jacek R. Wiśniewski 3, Diana Busch 4, Christian Gröer 4, Stefan Oswald 4, Agneta Norén 5, Frederik Weiss 6, Helen S. Hammer 6, Thomas O. Joos 6, Oliver Poetz 6, Brahim Achour 7, Amin Rostami-Hodjegan 7, Evita van de Steeg 8, Heleen M. Wortelboer 8, Per Artursson 1, * 1 Department of Pharmacy, Uppsala University, Uppsala Sweden 2 Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development Biotech Unit, AstraZeneca R&D, Mölndal 43150, Sweden 3 Biochemical Proteomics Group, Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried 82152, Germany 4 Center of Drug Absorption and Transport, Department of Clinical Pharmacology, University Medicine of Greifswald, Greifswald 17489, Germany 5 Department of Surgical Sciences, Uppsala University, Uppsala , Sweden 6 NMI Natural and Medical Sciences Institute, University of Tübingen, Reutlingen 72440, Germany 7 Centre for Applied Pharmacokinetic Research, University of Manchester, Manchester M13 9PL, United Kingdom 8 TNO (Netherlands Organization for Applied Scientific Research), 3700 AJ Zeist, Netherlands

2 Christine Wegler Supporting information 2 (13) Supporting information Methods Laboratory a. Targeted proteomics, whole cell lysates Tissue preparation The liver tissue samples (~100mg) were lysed and homogenized in 1ml lysis buffer on ice (100mM Tris/HCl, ph 7.8, 2w/v % SDS and 50mM DTT) with a homogenizer (IKA, T10 basic) for a maximum of 30 seconds. Proteins were denatured at 95 C for 5 min and DNA sheared with a rod sonicator (10 pulses during 20 seconds, 20 % amplitude) with the samples kept on ice. Total protein and peptide determination Total protein concentration of the lysate and peptide concentrations of the digests were determined with the tryptophan fluorescence method 1. Briefly, the tryptophan amount in the lysates and digests were determined by measuring fluorescence (excitation 295nm with 5nm band width, and emission 350nm with 10nm band width) in the samples and in tryptophan standard solutions using buffered urea. Sample preparation and digestion procedure Proteins were purified using the filter aided sample preparation (FASP)-method. Briefly, 100μg protein was depleted of SDS using a urea buffer (UA: 8M urea, 0.1M Tris/HCl ph 8.5) and alkylated with iodoacetamide (IAA, 0.05M in UA) for 20 min on YM-30 microcon filter units (Millipore). The proteins were digested on the filters with sequencing grade modified trypsin (1:40 enzyme/protein) for 16 hours at 37 C. The digested peptides were eluted using 2.5 % formic acid and their concentration was determined using the tryptophan fluorescence assay as described above. Digests were centrifugally evaporated and re-suspended in 50 % acetonitrile with 0.1 % formic acid to a final concentration of 0.25mg/ml total peptide. SIL peptide surrogates for the proteins to be quantified were spiked in the samples. LC-MS/MS analysis 2 μg total peptides were injected for each sample. Peptides were separated on a C18 column (Acquity UPLC BEH, 2.1 x 100mm, 1.7µm), with a gradient of mobile phase A (water, 0.1 % formic acid) and mobile phase B (acetonitrile, 0.1 % formic acid) using two methods. For the quantification of transporters, the peptides were eluted with a 13 minute gradient of 2-30 % mobile phase B. For enzymes a 13 minute linear gradient of 7-40 % mobile phase B was used followed by a 2 min wash out with 90 % mobile phase B at a flow rate of 500µl/min using an Agilent 1290 LC-system. Peptides were quantified using a QTRAP 6500 (AB Sciex) run in scheduled multiple reaction monitoring (MRM)- mode, with up to 84 simultaneously acquired transitions. Data acquisition was performed with a target scan time of 0.5 s and MRM detection window of 60 s. Three transitions per surrogate peptide were monitored for quantification MRM-mode. More detailed description of the parameters and criteria for quantification can be found in the supplementary excel file. Calibration curves were used for the enzymes (0.005, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10.0, 25.0, 50.0 nm) and transporters (0.005, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10.0, 25.0 nm) to quantify the proteins in the digests. Quality control (QC) samples with mixtures of the peptide standards were used (0.1, 1.0, 10.0 nm). Stable isotope-labelled internal standards were added to all standards and samples to a final concentration of 5 nm. Data analysis Data was processed using MultiQuant (Version , AB Sciex). Protein concentrations were calculated by the peak area ratios of the internal standard peptide and the sample peptide transitions. Final quantifications represent the sum of values of three transitions for each peptide. Limit of quantification was determined as the lowest quantifiable concentration (signal to noise > 10) of peptide standard for each protein. Laboratory b. Global proteomics, total protein approach Tissue preparation The tissue samples were homogenized with a homogenizer (IKA, Ultra Turrax T8) in a lysis buffer (20mM Tris-HCl, ph 7.4, 1mM EDTA, 1mM EGTA, 50mM DTT, 1mM PMSF, 2 % SDS). Proteins were denatured at 100 C for 5 min. Total protein and peptide determination Total protein and peptide concentration was determined with the tryptophan fluorescence method as described for laboratory a 1. Sample preparation and digestion procedures Protein lysates were processed on YM-30 microcon filter units (Millipore) using the multiple enzyme digestion-filter aided sample preparation (MED-FASP)

3 Christine Wegler Supporting information 3 (13) protocol using endoproteinase LysC and trypsin 2. Both digestion products were precleaned and fractionated using the pipette tip SAX (strong anion exchanger) method 3. Briefly, aliquots containing 16 µg of total peptide were diluted with 200 µl of the buffer, ph 12, containing 0.02 M acetic acid, 0.02 M phosphoric acid, and 0.02 M boronic acid sodium hydroxide and loaded onto the columns. The flowthrough fraction was discarded. Subsequently, the Lys C peptides were eluted with buffer solutions of ph 6 and 2 whereas the tryptic peptides were eluted in a single fraction with the buffer of ph 2. LC-MS/MS analysis 4 μg peptides were separated over a C 18 reverse phase column (50 cm long, 75 μm inner diameter, in-house packed with ReproSil-Pur C 18 -AQ 1.8 μm resin by Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) and were eluted with a linear gradient of 5 30 % buffer B (80 % acetonitrile and 0.5 % acetic acid) at a flow rate of 200 nl/min over 150 min. This was followed by 10 min elution from 30 to 60 % buffer B, a washout of 95 % buffer B and re-equilibration with buffer A. Peptides were electro-sprayed and analyzed on a Q Exactive HF mass spectrometer using a data-dependent mode with survey scans acquired at a resolution of 50,000 at m/z 200 (transient time 256 ms). Up to the top 15 most abundant isotope patterns with charge 2 from the survey scan were selected with an isolation window of 1.6 Th and fragmented by HCD with normalized collision energies of 25. The maximum ion injection times for the survey scan and the MS/MS scans were 20 ms and 60 ms, respectively. The ion target value for both scan modes were set to 10 6, meaning that most MS/MS scans accumulated ions to 60 ms. The dynamic exclusion was 25s and 10 ppm. Lysates of HeLa cells were used as quality control samples to ensure MS/MS efficiency. Data analysis The MS data were analyzed using the software environment MaxQuant ( version and its built-in Andromeda search engine. Proteins were identified by searching MS and MS/MS data against the complete human proteome sequences from UniProtKB, version of May 2013, containing 88,820 sequences, respectively. Carbamido-methylation of cysteine was set as fixed modification and N-terminal acetylation and oxidation of methionine as variable modifications. Up to two missed cleavages were allowed. The initial allowed mass deviation of the precursor ion was up to 6 ppm and up to 20 ppm for the fragment masses. Mass accuracy of the precursor ions was improved by time-dependent recalibration algorithms of MaxQuant. The match between runs option was enabled to match identifications across samples within a time window of 120 s of the aligned retention times. The maximum false peptide and protein discovery rates were set to Protein matching to the reverse database or identified only with modified peptides were filtered out. Protein titers were calculated using the Total Protein Approach (TPA) 4, using the relationship Protein concentration (i): C(i) = MS signal (i) Total MS signal MW(i) [ mol ] g total protein Laboratory c. Targeted proteomics, Immunoaffinity enrichment Tissue preparation Liver tissue was pulverized using a ball mill. A minimum of 10 mg tissue was required. Lysis buffer was added with the 10-fold volume (µl) of the weighed liver piece value (mg). Samples were incubated for 1 h at 4 C under continuous rotation. Total protein and peptide determination Protein concentrations were determined using a BCA assay kit (Thermo). Standard peptides (isotopically labeled) were quantified with amino acid analysis on an HPLC system. Digestion procedure Samples were diluted in triethanolamine (50 mm, ph 8.5) with 0.5 % NOG, reduced by TCEP (5 mm) for 5 min at 99 C, and alkylated by IAA (10 mm) for 30 min at RT. Trypsin was added in a ratio of 1:10 (trypsin:protein). After 16 h incubation at 37 C, additional trypsin (ratio 1:10) was added for further digestion up to 40 h. Peptide groups were enriched using an automatic immunoprecipitation procedure. Multi-specific antibodies (TXP antibodies) recognizing short common sequences at the C-term of proteotypic peptide fragments were mixed with isotopically labeled peptides and digested samples. Peptide-antibody-complexes were precipitated with magnetic protein G microspheres using a magnetic particle processor. Elution was done in 1 % formic acid.

4 Christine Wegler Supporting information 4 (13) LC-MS/MS analysis Peptides were desalted by a PepMap100 µ-precolumn (0.3 mm I.D. x 5 mm, Thermo) and separated by an Acclaim Rapid Separation LC (RSLC) Column (75 µm I.D. x 150 mm, Thermo) on an UltiMate 3000 RSLCnano LC system (Thermo). Mobile phase A was composed of ddh2o with 0.1 % TFA, mobile phase B of 80 % acetonitrile with 0.1 % formic acid. Transporters were eluted using a 2.75-minute gradient with % mobile phase B and a flow rate of 1000 nl/min. Enzymes were eluted using an 8-minute gradient with % mobile phase B and a flow rate of 300 nl/min. Peptides were quantified with a Q Exactive Plus (Thermo) using targeted Single- Ion-Monitoring (tsim). Data analysis Ion chromatograms were processed with Pinpoint 1.4 (Thermo). Concentrations were calculated by the peak area ratios of isotopically labeled and endogenous peptides on parent ion level. To avoid influences of non-labeled standard peptide impurities on the correctness of quantification the LLOQ was defined as 1 % of the spiked standard peptide amount. That is, LLOQ was defined as 0.05 fmol/µg for CYPs and fmol/µg for analyte-dependent transporters. More detailed description of the parameters and criteria for quantification can be found in the supplementary excel file. Laboratory d. Targeted proteomics, QconCat Tissue preparation Homogenization of tissue was carried out with a rotor-stator homogenizer (Dremel UK, Middlesex, UK) system in potassium phosphate buffer (250 mm, ph 7.25, 1.15 % KCl) on ice followed by sonication on ice using an Omni Ruptor 400 Ultrasonic homogeniser (Omni International, Kennesaw, GA). Membrane fractions were prepared by centrifugation at 10,000g for 20 min at 4 C, followed by ultracentrifugation at 100,000g for 75 min at 4 C. Total protein and peptide determination Total protein content was determined using a BCA assay done in triplicates using two different dilutions of membrane protein. Peptide recovery was determined after proteolysis using a gravimetric method 5. Sample preparation and digestion procedures Membrane fraction and QconCAT proteins were digested separately. Samples were digested as reported previously 5. The QconCAT was digested using Perfinity (now SMART digest, Thermo Fisher Scientific, Waltham, MA). Briefly, the 50 μl aliquots were diluted in Perfinity FLASH digestion buffer (1:4), then added to FLASH tubes, and placed in the thermomixer. Tubes were incubated for 75 minutes, at 70 C, with mixing at 1400 rpm. Following digestion, proteins were reduced with 10 mm DTT (1 h, 56 C), and subsequently alkylated with 15 mm IAA (45 min, RT). The digests were then acidified with formic acid (FA). The QconCAT digests were desalted using POROS R3 beads (Life Technologies, Paisley, UK). TELOS filtration microplate inserts (Kinesis Scientific Experts, St Neots, UK) were prepared by adding 1 mg of POROS R3 Reversed-Phase Media (Life Technologies, Paisley, UK). Beads were wet by the addition of 50 % (v/v) acetonitrile, and subsequently equilibrated with 0.1 % (v/v) formic acid. The standards were applied and washed twice with 0.1 % (v/v) formic acid followed by elution in 50 % (v/v) acetonitrile, 0.1 % (v/v) formic acid and complete evaporation by vacuum centrifugation. The sample digests were desalted using an in-house 96- well format SPE. Desalting plates were generated by the addition of 1 mg OLIGO R3 beads (Life Technologies, Paisley, UK) to each well of a Corning 96-well plate containing a 0.2 µm PVDF membrane (Fisher Scientific, Loughborough, UK). Beads were firstly wet by the addition of 50 % (v/v) acetonitrile and equilibrated using 0.1 % (v/v) formic acid. The sample digests were applied and then washed twice with 0.1 % (v/v) formic acid. Each time the liquid was removed by centrifugation (1 min, 1400 rpm). Samples were eluted in 50 % (v/v) acetonitrile, 0.1 % (v/v) formic acid, collected via centrifugation (1 min, 1400 rpm) and completely evaporated by vacuum centrifugation. The desalted QconCAT digests and sample digests were combined (1:10 QconCAT). QconCAT was quantified separately using a light NNOP (non-naturally occurring peptide) standard (1:1). QconCAT labeling was carried out by expression using E. coli cultured in 13 C-arginine and lysine heavy isotope enriched minimum medium 6. LC-MS/MS-analysis The digests were analyzed by MRM-LC-MS/MS using an UltiMate 3000 Rapid Separation LC (RSLC, Dionex Corporation, Sunnyvale, CA) coupled to a QTRAP 6500 (AB Sciex, Framingham, MA) mass spectrometer operated using Analyst 1.6 software (AB Sciex). 1 µl of each peptide mixture was separated using a gradient from 95 % A (0.1 % FA in water) and 5 % B (0.1 % FA in

5 Christine Wegler Supporting information 5 (13) acetonitrile) to 30 % B, in 46 min at 300 nl min -1, using a 250 mm x 75 μm i.d. 1.7 µm CSH130 C18, analytical column (Waters, Milford, MA). The transition schedules were selected using Skyline (version , MacCross Lab Software). For each transition the dwell time was 10 ms and cycle time s. Quantification of the QconCAT using the NNOP standard was confirmed using Orbitrap Elite (Thermo Fisher Scientific, Waltham, MA). Data analysis The ratios of native (light) and standard (heavy) selected transitions for each peptide were calculated on Skyline. The lower limit of quantification was determined previously (0.1 fmol/µg protein) using a cut-off intensity value of 3000 arbitrary units and reproducibility of the quantitative signal (<15 % CV) between quality control runs. Enzyme/transporter abundances (in fmol/µg cell fraction protein) were determined from QconCAT-derived proteotypic peptide standards as reported previously 5. More detailed description of the parameters and criteria for quantification can be found in the supplementary excel file. Laboratory e. Targeted proteomics, microsomal fractionation and membrane enrichment Tissue preparation Liver samples were stored at -80 C until processing. All pipetting steps were performed in Protein LoBind Tubes (Eppendorf) with UltraFine TM siliconized tips (VWR). Liver tissue samples were disrupted to a tissue powder using cell crusher paddles pre-cooled in liquid nitrogen, and then prepared for quantification of either enzymes or transporters 7, 8. Sample preparation For enzyme quantification approximately 300 mg of the tissue powder was homogenized in lysis buffer (0.2 % SDS, 5 mm EDTA, 5 µl/ml protease inhibitor) using a douncer. After incubation for 30 min (40 rpm at 4 C) samples were centrifuged with 600 g for 5 min at 4 C. The resulting supernatant was subjected to ultra-centrifugation for 70 min (100,000 g, 4 C). The obtained pellet was washed with a buffer containing potassium dihydrogen phosphate (10 mm), EDTA (1 mm) and protease inhibitor (5 µg/ml) followed by 70 min of ultracentrifugation (100,000 g, 4 C) and resuspension of the pellet in a buffer containing sucrose (0.25 M), EDTA (1 mm) and protease inhibitor (5µg/ml). Subsequently, samples were centrifuged with 16,000 g at 4 C for 15 min. To isolate crude membranes and enrich for transporters the ProteoExtract Native Membrane Protein Extraction Kit (Merck) was used according to the manufacturer s instructions with some modifications. Briefly, approximately 80 mg of tissue powder was mixed with 700 µl cold extraction buffer I (containing 5 µl/ml protease inhibitor) and incubated for 15 min with 40 rpm at 4 C. After a centrifugation (15 min, 16,000g, 4 C) the supernatant was discarded and the pellet was re-suspended in 300 µl cold extraction buffer II (containing 5 µl/ml protease inhibitor). The samples were incubated for 1 h at 4 C with 40 rpm followed by 15 min centrifugation with 16,000g at 4 C. Total protein and peptide determination Total amount of protein in the cell fraction was determined using the BCA-assay using BSA as standard. The concentration of the extracted proteins was adjusted to 2 mg/ml. Digestion procedure 100 µl of each sample was mixed with 50 µl ammonium hydrogen carbonate (50 mm, ph 7.8) or 40 µl ammonium hydrogen carbonate (50 mm, ph 7.8) and 10 µl ProteaseMAX TM (Promega) for microsomal and crude membrane fraction, respectively. Preparations were reduced with 10 µl DTT (200 mm) for 20 min at 60 C, followed by alkylation with 10 µl IAA (400 mm) for 15 min at 37 C. The proteins were finally digested with trypsin (1:40 enzyme/protein) for 16 h at 37 C. The reaction was stopped by addition of 20 µl formic acid (10 % v/v). After 15 min of centrifugation (16,000 g, 4 C) stable isotope-labelled internal standards were added to each sample (final concentration of 5 nm). Preparation of calibration curves and quality controls Working solutions of the light peptides were prepared in acetonitrile (0.1 % formic acid) and water (0.1 % formic acid) (50:50, v/v). Working solutions of the heavy peptides were prepared in acetonitrile (0.1 % formic acid) and water (0.1 % formic acid) (90:10, v/v). Calibration curves and quality control (QC) samples were prepared by spiking blank matrix (50 mm Ammoniumbicarbonate, ph 7.8) with increasing amounts of each peptide to generate the following target concentrations for transporters (calibration curve: 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10.0 nm, quality control (QC) samples: 0.1, 1.0, 10.0 nm) and enzymes (calibration curve: 0.1, 0.25, 0.5, 1.0,

6 Christine Wegler Supporting information 6 (13) 2.5, 5.0, 10.0, 25.0, 50.0 nm, quality control (QC) samples: 0.5, 5.0, 50.0 nm), respectively. The stable isotope-labelled internal standards were added to all samples (final concentration: 5 nm). LC-MS/MS analysis The peptides were separated after injection of 15 µl sample volume over an Accucore C 18 reverse phase column (100 mm long, 3.0 mm inner diameter, ThermoFisher) and eluted at a flow rate of 500 µl/min using acetonitrile with 0.1 % FA (solvent A) as well as water with 0.1 % FA (solvent B) with a linear gradient of 2 25 % solvent A over 45 min followed by a 4 minute washout with 55 % solvent A. The QTRAP 5500 mass spectrometer (AB Sciex) operated with the electrospray ionization (ESI) TurboIon interface and in the positive ion mode to monitor the m/z transitions for all peptides and their internal standards. For each peptide, three MRMs were simultaneously monitored. A scheduled MRM acquisition method was constructed using manually optimized de-clustering potentials, collision energies, and collision cell entrance and exit potentials. The following ion source parameters were applied: 4.5 kv spray voltage, 500 C source temperature, curtain gas: 50 psi, gas 1: 50 psi, gas 2: 50 psi and CAD gas: HIGH (all nitrogen). Data analysis All chromatograms were evaluated with the Analyst 1.6 software (AB Sciex) using the internal standard method and peak-area ratios for calculation (linear regression, 1/x weighting). Final quantifications represent mean values of three transitions for each peptide. More detailed description of the parameters and criteria for quantification can be found in the supplementary excel file. Laboratory f. Targeted proteomics, membrane enrichment Tissue preparation Liver tissues were homogenized in hypotonic buffer (0.5 mm Sodium phosphate, 0.1 mm EDTA, 0.1 mm protease inhibitors containing phenylmethylsulfonylfluoride, aprotinin, leupeptin, and pepstatin). The homogenized tissue was centrifuged at 100,000 g for 30 min at 4 C using a LE-80k Centrifuge with SW rotor (Beckman Counter,USA). The resulting pellet was re-suspended in hypotonic buffer and further suspended in isotonic buffer (10mM Tris-Hepes and 250 mm sucrose, ph 7.4). The extract was homogenized and centrifuged again (8,000 x g for 10 min at 4 C). The supernatant was collected and ultracentrifuged (100,000 x g for 30 min at 4 C). The obtained pellet was resuspended in isotonic buffer, layered on top of 38 % sucrose solution and centrifuged at 100,000 x g for 90 min at 4 C. The plasma membrane (turbid layer at the interface) was recovered, diluted with isotonic buffer and further centrifuged (100,000 x g for 40 min at 4 C). The resulting pellet, containing the plasma membrane, was resuspended in 50 µl isotonic buffer. Protein levels in the membrane fractions were measured using the Bradford method (Biorad, USA). Plasma membrane extracts (~100 µg) were diluted with 2 volumes of 90 % methanol. The proteins were reduced with DTT (0.01 M) for 60 min at 37 C followed by alkylation with IAA (0.04 M) for 20 min at RT in the dark. Digestion was performed overnight by the addition of calcium chloride (final concentration 1 nm) and 2.5 µg trypsin (1:40 enzyme:protein) (Promega) in 17 % methanol by diluting the solution with 50 mm ammonium bicarbonate, and further incubated for another 2 h with additional 2.5 µg trypsin. The protein digests were centrifugally evaporated and dissolved in 15 % acetonitrile, 0.1 % formic acid and mixed with 5 ng/ml internal standard labeled with 15N and 13C. LC-MS/MS analysis Peptides were separated on a C 18 -column (Acquity BEH UPLC column, 2.1 x 100 mm, inner diameter 1.7µm) using a linear gradient of 5-45 % mobile phase B (acetonitrile with 0.1 % formic acid) for 5 min with a flow of 600 µl/min, followed by a 2 min wash-out with 100 % mobile phase B. Peptides were ionized with electrospray and quantification was performed with a 6500 QTrap (ABSciex) using a scheduled MRM-mode. Three transitions were monitored for each peptide. Data analysis Peak identification and quantification was performed using Analyst software version 1.6. The protein concentrations were calculated by the peak area ratios of the internal standard peptide and the sample peptide transitions. Final quantifications represent mean values of three transitions for each peptide. The lower limit of quantification was determined by the lowest accurate concentration of the calibration curve ( 15 % deviation). More detailed description of the parameters and criteria for quantification can be found in the supplementary excel file.

7 Christine Wegler Supporting information 7 (13) Intra-laboratory reproducibility Each laboratory received duplicate tissue samples from the same ten individuals. Tissue preparation and analysis were performed at two independent occasions. The protein concentrations obtained from each run were compared to measure the within-laboratory accuracy and precision. The average fold error (AFE) and absolute average fold error (AAFE), were calculated according to equation 1 and 2 9, respectively. Equation 1: AFE = 10 log (fold error) N log (fold error) Equation 2: AAFE = 10 N where the fold-error is the ratio between the protein concentrations obtained from the independent runs, and N denotes the number of protein measurements included in the analysis. AFE is the geometric mean of the fold-errors and gives a measure of the overall bias (difference from the reference value at 1) towards one of the runs, while AAFE gives an estimate of the spread from the reference value at 1 9. Results Human liver tissue Human liver tissue samples were prepared and analyzed using the respective in-house methodologies. Preparation of whole tissue lysates (Lab a, b and c) only required 10 mg tissue while up to 530 mg ( mg) tissue was required for the various cell fractionation procedures. An average of the actual amounts of tissue processed, from the ten individuals, in the different laboratories can be found in Figure S1. Influence of peptide selection on quantification The use of different proteotypic peptides can influence protein quantification 10, 11. However, in our study, we could not observe this effect (Figure 5f). We therefore further investigated this issue by comparing all proteins quantified with matching peptides in the different laboratories. In Figure S4, the fractions of matching peptides across the laboratories are shown (intersections of the circles; for peptide sequences see Table S3). Surprisingly, the correlations between laboratories with many overlapping peptides (e.g. Lab a - Lab b, Lab a - Lab e, and Lab b - Lab e, Figure S4), and laboratories with few matching peptides (e.g. Lab a - Lab c) were comparable. Further, the lower protein concentrations observed after subcellular fractionation procedures could not be explained by differences in fraction of matching peptides. For instance, between Lab a (whole tissue digests) and Lab e (crude membrane fractions) better correlation was obtained for proteins quantified with matched peptides (r = 0.8) than unmatched peptides (r = 0.54). However, all protein levels were on average 3-fold lower in Lab e.

8 Christine Wegler Supporting information 8 (13) Supplementary figures and tables Figure S1. Amount of tissue processed by the different laboratories, where Labs a, b and c prepared whole tissue lysates while Labs d, e and f isolated microsomal/crude membrane and plasma membrane fractions, respectively. Figure S2. Protein levels of drug transporters in microsomal fractions from the literature meta-analyses 12-14, and unscaled protein levels from crude membrane fractions (Labs d and e) and plasma membrane fractions (Lab f) (a). Transporter protein levels obtained in whole tissue digests (Labs a, b and c) and in subcellular fractions (Labs d, e and f) reveal low enrichment after subcellular fractionation (b).

9 Christine Wegler Supporting information 9 (13) Figure S3. Pearson product-moment correlation coefficients (r) of the ten liver samples, between the laboratories. Proteins with correlated sample orders (r 0.79) between laboratories are marked with green.

10 Christine Wegler Supporting information 10 (13) Figure S4. Influence of reference peptide selection on end-point protein quantification. To investigate the impact of peptide selection, protein levels obtained by matching peptide standards across laboratories (red) were selected for comparison. The number of proteins quantified with the same peptides is shown as intersections of circles for each lab-lab comparison. r Pearson s correlation coefficient.

11 Christine Wegler Supporting information 11 (13) Figure S5. Prediction of hepatic intrinsic uptake clearance (CL int,uptake ) of atorvastatin. A static mathematical model 15 was used in predicting the influence of SLCO1B1, SLCO2B1 and SLCO1B3 abundance on atorvastatin clearance. Predicted CL int,uptake of atorvastatin by the three transporters, respectively, in the ten liver samples based on the protein levels (fmol/µg total protein) quantified in the six laboratories. In Labs c and d only SLCO1B3 and SLCO1B1 was quantified, respectively. The dashed line represents geometrical mean values from the ten individuals. Table S1. Demographic data for human liver tissue donors. Individual Gender Age Diagnosis 1 M 54 CRC 2 M 79 CRC 3 F 49 CRC 4 M 62 CRC 5 F 73 CRC 6 M 51 CRC 7 F 77 CRC 8 M 72 CRC 9 M 58 CRC 10 M 75 HCC M: male, F: female, CRC: colorectal cancer, HCC: hepatocellular carcinoma

12 Christine Wegler Supporting information 12 (13) Table S2. Proteins relevant to drug disposition included in the study. Proteins marked X were quantified in the laboratories. Gene Protein Lab a Lab b Lab c Lab d Lab e Lab f ABCB1 MDR-1/Pgp X X X X X X ABCC2 MRP2 X X NA X X X ABCC3 MRP3 X X NA X X X ABCG2 BCRP X X NA X X X SLC22A1 OCT1 X X X X X X SLC47A1 MATE1 X X NA X X NA SLCO1A2 OATP1A2 X X NA X X NA SLCO1B1 OATP1B1 X X NA X X X SLCO1B3 OATP1B3 X X X X X X SLCO2B1 OATP2B1 X X NA X X X CYP1A2 CYP1A2 X X X NA X NA CYP2B6 CYP2B6 X X X NA X NA CYP2C8 CYP2C8 X X X NA X NA CYP2C9 CYP2C9 X X X NA X NA CYP2C19 CYP2C19 X X X NA X NA CYP2D6 CYP2D6 X X X NA X NA CYP2E1 CYP2E1 X X X NA X NA CYP3A4 CYP3A4 X X X NA X NA CYP3A5 CYP3A5 X X X NA X NA UGT1A1 UGT1A1 X X NA NA X NA UGT1A3 UGT1A3 X X NA NA X NA UGT2B7 UGT2B7 X X NA NA X NA UGT2B15 UGT2B15 X X NA NA X NA X: Quantification method available for the protein NA: In-house method not available

13 Christine Wegler Supporting information 13 (13) Table S3. Surrogate peptides used for quantification of the different proteins in the respective laboratory. Lab b 1 Gene Lab a Lab c Lab d Lab e Lab f [*] ABCB1 AGAVAEEVLAAIR [*] 25 AGAVAEEVLAAIR IATEAIENFR NTTGALTTR EANIHAFIESLPNK EANIHAFIESLPNK ABCC2 LTIIPQDPILFSGSLR [*] 24 LVNDIFTFVSPQLLK LTIIPQDPILFSGSLR [*] VLGPNGLLK ABCC3 IDGLNVADIGLHDLR [*] 21 SPIYSHFSETVTGASVIR [*] IDGLNVADIGLHDLR [*] ALVITNSVK ABCG2 SSLLDVLAAR 5 VIQELGLDK SSLLDVLAAR SSLLDVLAAR SLC22A1 ENTIYLK [*] 5 LSPSFADLFR [*] MLSLEEDVTEK ENTIYLK [*] LPPADLK SLC47A1 GGPEATLEVR 2 HVGVILQR GGPEATLEVR DHVGYIFTTDR SLCO1A2 EGLETNADIIK 0 EGLETNADIIK EGLETNADIIK IYDSTTFR SLCO1B1 NVTGFFQSFK [*] 12 YVEQQYGQPSSK [*] NVTGFFQSFK[*] LNTVGIAK IYNSVFFGR SLCO1B3 NVTGFFQSLK 5 IYNSVFFGR [*] TLGGILAPIYFGALIDK [*] SLCO2B1 SSPAVEQQLLVSGPGK [*] 3 SSPAVEQQLLVSGPGK [*] CYP1A2 YLPNPALQR 23 ELDTVIGR [*] YLPNPALQR CYP2B6 TEAFIPFSLGK [*] 18 AEAFSGR TEAFIPFSLGK [*] CYP2C8 VQEEIDHVIGR [*] 21 EALIDNGEEFSGR [*] VQEEIDHVIGR [*] CYP2C9 GIFPLAER [*] 14 GIFPLAER [*] GIFPLAER [*] CYP2C19 GHFPLAER [*] 12 GHFPLAER [*] GHFPLAER [*] CYP2D6 DIEVQGFR 20 GTTLITNLSSVLK [*] DIEVQGFR CYP2E1 FITLVPSNLPHEATR [*] 35 DEFSGR FITLVPSNLPHEATR [*] CYP3A4 EVTNFLR [*] 9 LQEEIDAVLPNK EVTNFLR [*] CYP3A5 DTINFLSK [*] 10 EIDAVLPNK DTINFLSK [*] UGT1A1 TYPVPFQR 8 TYPVPFQR UGT1A3 YLSIPTVFFLR [*] 7 YLSIPTVFFLR [*] UGT2B7 IEIYPTSLTK [*] 17 IEIYPTSLTK [*] UGT2B15 SVINDPVYK [*] 9 SVINDPVYK [*] SSISTVEK 1 Number of unique and razor peptides attributed to a protein. In blue: Matching peptides in targeted proteomics approaches, [*] in red: Surrogate peptide was found in the peptides used for quantification in the global proteomics approach

14 Christine Wegler Supporting information 1 (1) Table S4. Comparison of methodologies applied in the different laboratories Lab a Lab b Lab c Lab d Lab e Lab f Cell fraction Whole tissue lysate Whole tissue lysate Whole tissue lysate Microsomes/ crude membrane Microsomes/ Crude membrane Plasma membrane Solubilization SDS 2 % SDS 2 % Triton 0.5 % SDS 0.01 % Protein extraction Protein digestion FASP Trypsin (1:40) 16h MED-FASP LysC (1:50) 18h Trypsin (1:50) 3h Trypsin (1:10) 16h Trypsin (1:10) 24h DOC 10 % SDS 0.2 % LysC (1:50) 4h Trypsin (1:20) 18h Trypsin (1:40) 16h Trypsin (1:200) 16h Trypsin (1:200) 2h Peptide enrichment Total protein content Immunoprecipitation Tryptophan Tryptophan BCA BCA BCA Bradford method Peptide content LC-MS/MS analysis Tryptophan MRM, SIL Tryptophan Label free, Total Protein Approach Gravimetric method tsim, SIL MRM, QconCAT MRM, SIL MRM, SIL SDS sodium dodecyl sulfate, DOC deoxycholate, SIL peptides Stable isotope labeled peptides, MRM multiple reaction monitoring, tsim targeted single ion monitoring, QconQAT quantification concatemer, MED-FASP multi enzyme digestion filter aided sample preparation, BCA bicinchoninic acid assay

15 Christine Wegler Supporting information 1 (2) Table S5. Protein concentrations (fmol/µg total protein) obtained using the six methodologies. Protein concentrations from ten individuals are displayed as geometrical mean and range. CV% was calculated for protein levels across all labs, for each protein. Gene Lab a Geomean Lab a Range Lab b Geomean Lab b Range Lab c Geomean Lab c Range Lab d Geomean Lab d Range Lab e Geomean Lab e Range Lab f Geomean Lab f Range CV% all Labs ABCB ABCC ABCC ABCG SLC22A SLC47A SLCO1A SLCO1B SLCO1B SLCO2B CYP1A CYP2B CYP2C CYP2C CYP2C CYP2D CYP2E CYP3A CYP3A UGT1A UGT1A UGT2B UGT2B Unit: fmol/µg total protein

16 Christine Wegler Supporting information 2 (2) Table S6. P-values for comparisons of mean protein levels between laboratories, calculated by Kruskal-Wallis test followed by Dunn's multiple comparisons test. Lab a vs Lab b Lab a vs Lab c Lab a vs Lab d Lab a vs Lab e Lab a vs Lab f Lab b vs Lab c Lab b vs Lab d Lab b vs Lab e Lab b vs Lab f Lab c vs Lab d Lab c vs Lab e Lab c vs Lab f Lab d vs Lab e Lab d vs Lab f ABCB1 ns p<0.05 ns ns p<0.001 p< ns p<0.05 p< ns ns ns ns p<0.01 p<0.05 ABCC2 ns ns ns p< ns ns p<0.001 p<0.05 ns p< ABCC3 ns ns ns p<0.001 ns p<0.01 p< ns p<0.05 ns ABCG2 p<0.001 p<0.01 ns ns p<0.001 p<0.01 SLC22A1 ns p< ns ns p< p<0.05 ns ns p< ns ns ns ns p<0.05 p<0.01 SLC47A1 p< SLCO1A2 SLCO1B1 ns ns p<0.001 p< ns ns p<0.001 ns p<0.01 ns SLCO1B3 p<0.01 ns p<0.05 p< p<0.05 ns ns ns p< ns SLCO2B1 p<0.01 ns p<0.05 p< p<0.05 ns ns ns p< ns CYP1A2 ns ns p<0.01 ns p< p<0.01 CYP2B6 p< p<0.05 ns ns p< p<0.05 CYP2C8 p<0.05 ns ns ns p< p<0.01 CYP2C9 ns ns p< ns p<0.01 p<0.05 CYP2C19 ns ns p<0.01 ns p<0.05 p<0.05 CYP2D6 ns ns ns ns p<0.001 p<0.05 CYP2E1 ns ns p<0.001 p<0.01 p< ns CYP3A4 ns ns p<0.01 ns p< p<0.05 CYP3A5 ns ns p< ns p<0.001 p<0.05 UGT1A1 p<0.01 ns p<0.01 UGT1A3 ns ns ns UGT2B7 ns p< p<0.05 UGT2B15 ns p<0.01 p< Wilcoxon matched-pairs signed rank test, tw o-tailed ns - nonsignificant difference in mean levels (10 individuals) of protein between laboratories Lab e vs Lab f

17 Christine Wegler Supporting information 1 (1) References 1. Wisniewski, J. R.; Gaugaz, F. Z. Fast and sensitive total protein and Peptide assays for proteomic analysis. Analytical chemistry 2015, 87, (8), Wisniewski, J. R.; Mann, M. Consecutive proteolytic digestion in an enzyme reactor increases depth of proteomic and phosphoproteomic analysis. Analytical chemistry 2012, 84, (6), Wisniewski, J. R.; Zougman, A.; Mann, M. Combination of FASP and StageTip-based fractionation allows in-depth analysis of the hippocampal membrane proteome. Journal of proteome research 2009, 8, (12), Wisniewski, J. R.; Rakus, D. Multi-enzyme digestion FASP and the 'Total Protein Approach'-based absolute quantification of the Escherichia coli proteome. Journal of proteomics 2014, 109, Harwood, M. D.; Achour, B.; Russell, M. R.; Carlson, G. L.; Warhurst, G.; Rostami- Hodjegan, A. Application of an LC-MS/MS method for the simultaneous quantification of human intestinal transporter proteins absolute abundance using a QconCAT technique. Journal of pharmaceutical and biomedical analysis 2015, 110, Russell, M. R.; Achour, B.; McKenzie, E. A.; Lopez, R.; Harwood, M. D.; Rostami- Hodjegan, A.; Barber, J. Alternative fusion protein strategies to express recalcitrant QconCAT proteins for quantitative proteomics of human drug metabolizing enzymes and transporters. Journal of proteome research 2013, 12, (12), Groer, C.; Bruck, S.; Lai, Y.; Paulick, A.; Busemann, A.; Heidecke, C. D.; Siegmund, W.; Oswald, S. LC-MS/MS-based quantification of clinically relevant intestinal uptake and efflux transporter proteins. Journal of pharmaceutical and biomedical analysis 2013, 85, Groer, C.; Busch, D.; Patrzyk, M.; Beyer, K.; Busemann, A.; Heidecke, C. D.; Drozdzik, M.; Siegmund, W.; Oswald, S. Absolute protein quantification of clinically relevant cytochrome P450 enzymes and UDP-glucuronosyltransferases by mass spectrometry-based targeted proteomics. Journal of pharmaceutical and biomedical analysis 2014, 100, Tang, H.; Hussain, A.; Leal, M.; Mayersohn, M.; Fluhler, E. Interspecies prediction of human drug clearance based on scaling data from one or two animal species. Drug metabolism and disposition: the biological fate of chemicals 2007, 35, (10), Pan, S.; Aebersold, R.; Chen, R.; Rush, J.; Goodlett, D. R.; McIntosh, M. W.; Zhang, J.; Brentnall, T. A. Mass spectrometry based targeted protein quantification: methods and applications. Journal of proteome research 2009, 8, (2), Prasad, B.; Evers, R.; Gupta, A.; Hop, C. E.; Salphati, L.; Shukla, S.; Ambudkar, S. V.; Unadkat, J. D. Interindividual variability in hepatic organic anion-transporting polypeptides and P- glycoprotein (ABCB1) protein expression: quantification by liquid chromatography tandem mass spectroscopy and influence of genotype, age, and sex. Drug metabolism and disposition: the biological fate of chemicals 2014, 42, (1), Achour, B.; Barber, J.; Rostami-Hodjegan, A. Expression of hepatic drug-metabolizing cytochrome p450 enzymes and their intercorrelations: a meta-analysis. Drug metabolism and disposition: the biological fate of chemicals 2014, 42, (8), Achour, B.; Rostami-Hodjegan, A.; Barber, J. Protein expression of various hepatic uridine 5'-diphosphate glucuronosyltransferase (UGT) enzymes and their inter-correlations: a metaanalysis. Biopharmaceutics & drug disposition 2014, 35, (6), Vildhede, A.; Wisniewski, J. R.; Noren, A.; Karlgren, M.; Artursson, P. Comparative Proteomic Analysis of Human Liver Tissue and Isolated Hepatocytes with a Focus on Proteins Determining Drug Exposure. Journal of proteome research 2015, 14, (8), Karlgren, M.; Vildhede, A.; Norinder, U.; Wisniewski, J. R.; Kimoto, E.; Lai, Y.; Haglund, U.; Artursson, P. Classification of inhibitors of hepatic organic anion transporting polypeptides (OATPs): influence of protein expression on drug-drug interactions. Journal of medicinal chemistry 2012, 55, (10),