Comparison of Protein Expressions of Drug-Metabolizing Enzymes between Human Liver and the Hepatic Cell Lines HepG2, Hep3B and Huh7 using SWATH and MRM-HR Proteomics
Human hepatic cell lines have been widely used as an in vitro model for the study of drug metabolism and liver toxicity. However, the validity of this model is still a subject of debate because the expressions of various proteins including drug-metabolizing enzymes (DMEs) in the cell lines can differ significantly from that of human livers. In the present study, we first conducted an untargeted proteomics of the microsomes of the cell lines HepG2, Hep3B, and Huh7 in comparison with human livers using a SWATH method. Furthermore, a targeted proteomic approach, named high-resolution multiple reaction monitoring (MRM-HR), was utilized to compare the expressions of pre-selected DMEs between human livers and the cell lines.
Sample Processing Protocol
Protein digestion was conducted according to a previously reported Lys-C/Trypsin combinatorial digestion protocol with some modifications. An aliquot of 100 μg protein of microsomes was mixed with the internal standard 0.5 μg BSA in an Eppendorf Protein LoBind tube. A 10-fold volume of pre-cooled acetone was added. The mixture was briefly vortexed and incubated at -20 °C for at least 2 hours, followed by centrifugation at 17,000 g for 15 min at 4 °C. The supernatants were discarded, then the precipitated proteins were added with 200 μL ice-cold 80% ethanol for a washing step. The mixture was centrifuged again at 17,000 g for 15 min at 4 °C. The supernatants were removed and the precipitated proteins were air-dried at room temperature. The dried proteins were resuspended in 100 μL of freshly prepared 4 mM dithiothreitol in 8 M urea solution containing 100 mM ammonium bicarbonate. Samples were briefly vortexed and sonicated, then incubated at 37 °C for 45 min. After samples were cooled down to room temperature, 100 μL of 20 mM iodoacetamide freshly prepared in 8 M urea/100 mM ammonium bicarbonate solution was added. The mixture was incubated at room temperature for 30 min in the dark for alkylation. Following the incubation, urea concentration was adjusted to 6 M by adding 50 mM ammonium bicarbonate, and lysyl endopeptidase was added for the first digestion step (protein to lysyl endopeptidase ratio = 100:1) at 37 °C for 6 h. Samples were then diluted to 1.6 M urea with 50 mM ammonium bicarbonate, followed by the second step of digestion with trypsin at a protein to trypsin ratio of 50:1 for an overnight incubation at 37 °C. The digestion was terminated by the addition of 1 μL trifluoroacetic acid. Digested peptides were extracted and purified using Waters Oasis HLB columns according to the manufacturer’s instructions. Peptides eluted from the columns were dried in a SpeedVac concentrator SPD1010, and reconstituted in 80 μL of 3% acetonitrile solution with 0.1% formic acid. The peptide samples were centrifuged at 17,000 g for 10 min at 4 °C, and half of the supernatant was collected and supplemented with 1 μL of the synthetic iRT standards solution from Biognosys AG prior to LC-MS/MS analysis.
Data Processing Protocol
IDA data were searched using MaxQuant software (version 22.214.171.124, Max Planck Institute of Biochemistry, Germany) with default settings. The human proteome fasta file with 20,237 protein entries downloaded from Uniport on 3/2/2017 was used as the reference sequences for the search. For the untargeted analysis, the Spectronaut software (version 11.0.15038.8, Biognosys AG, Schlieren, Switzerland) was used to process the SWATH data with the reference spectral library generated from the IDA searches. The SWATH data were also analyzed using the Skyline-daily program (version 126.96.36.19971, University of Washington, Seattle, WA) for the drug-metabolizing enzymes quantification. MRM-HR data were analyzed by Skyline-daily which automatically detects and matches the MS/MS chromatographic peaks against the spectral library generated from the IDA searches.
Shi J, Wang X, Lyu L, Jiang H, Zhu HJ. Comparison of protein expression between human livers and the hepatic cell lines HepG2, Hep3B, and Huh7 using SWATH and MRM-HR proteomics: Focusing on drug-metabolizing enzymes. Drug Metab Pharmacokinet. 2018 33(2):133-140 PubMed: 29610054