Validation of the Sample Preparation Protocol Based on 1DE-gel Concentration for Proteomic Analysis of HepaRG Cell Culture

Abstract

HepaRG is a promising cell line for the study of drug metabolism, which requires an integrated approach that includes genomics, transcriptomics, and proteomics. The genome and transcriptome of HepaRG have been studied quite well, while the proteome remains poorly characterized, which may be due, in particular, to the lack of a standardized sample preparation method for proteomic analysis. In this study, two methods of sample preparation for MS analysis of HepaRG cells were compared: protein extraction with RIPA buffer based on sodium dodecyl sulfate followed by 1DE-gel concentration and trypsinolysis in the gel (protocol 1), protein extraction with urea/thiourea buffer followed by trypsinolysis in solution (protocol 2). The efficacy of trypsinolysis was evaluated using an external standard, recombinant cytochrome P450 BM3 from the bacterium Bacillus megaterium. The search for peptides and proteins was carried out using the domestic proteomic machine IdentiProt. Trypsinolysis in gel (Protocol 1) allowed the identification of 82.7 ± 1.5 BM-3 peptides, whereas trypsinolysis in solution identified 76.0 ± 0.0 peptides, which averaged 49.8% and 45.8% of the theoretically possible amount, respectively. Both methods demonstrated a high correlation (r = 0.84) between the intensity values of the reported BM3 peptides. 8487 + 235 peptides and 1242 + 22 HepaRG proteins (protocol 1), 9415 + 276 peptides and 1478 + 34 proteins (protocol 2) were identified. Analysis of the identified proteins showed that both protocols made it possible to identify proteins in the molecular weight range of 6–629 kDa, with the majority of proteins (75%) in both cases being represented by low molecular weight proteins (10–100 kDa). The analysis of the protein composition of HepaRG cells made it possible to identify a greater number of membrane proteins, liver cell proteins and drug metabolism proteins for the sample preparation protocol using urea-based buffer, while the sample preparation protocol based on RIPA buffer solubilization with 1DE-gel concentration and trypsinolysis in the gel made it possible to identify a greater number of proteins associated with biological processes specific to the liver. Thus, the method of sample preparation based on 1DE-gel concentration and trypsinolysis in the gel has shown its effectiveness for characterizing the proteome of human hepatoma HepaRG cells and searching for proteins involved in the metabolism of xenobiotics and drugs.