The validation of sample preparation protocols for MS-based proteins detection in HepaRG cells

The protocols of sample preparation of human hepatoma cells HepaRG have been compared in terms of assessing the depth of panoramic proteomic analysis. The following sample preparation techniques were tested: 1 – solubilization of HepaRG cell proteins using a RIPA buffer, removal of detergents using 1DE-gel concentration procedure (modified SDS-PAGE without fractionation in a separating gel), in-gel trypsin digestion; 2 - lysis of cells with a solution containing urea and thiourea, in-solution tryptic digestion. The trypsin:protein ratio was 1:50 for each method of tryptic digestion of proteins from HepaRG cell extract. To compare the depth of the proteome covering under different sample preparation conditions, an external standard was introduced into protein extracts – recombinant cytochrome P450 BM-3 (Bifunctional cytochrome P450/NADPH-P450 reductase, cyp102A1) from the gram-positive bacterium Bacillus Megaterium, which does not share peptides with HepaRG proteins. Mass spectrometry analysis was performed using a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific). The identification of peptides and proteins from acquired MS/MS spectra was carried out using the IdentiProt proteomic search engine based on the IdentiPy algorithm. The assessment of sample preparation protocols was based on the number of identified cytochrome P450 BM-3 peptides in a complex mixture with peptides of HepaRG cell proteins. The efficiency of in-gel and in-solution digestion can be estimated by the number of BM-3 identified peptides compared with the theoretical number of all its possible peptides. Theoretical peptides from cytochrome P450 BM-3 was performed using an Expasy server. There were 166 theoretical tryptic peptides for BM-3 containing ≥ 7 aa (with taking into account complete hydrolytic cleavage and mis cleavage 1). The number of identified BM-3 peptides for method 1 was 82.7 ± 1.5 and method 2 was 76.0 ± 0, which averaged 49.8% and 45.8% of the theoretically possible amounts, respectively. The observed differences in the number of identified peptides using the tested protocols are statistically significant (p<0.05). Thus, the results obtained indicate that the sample preparation protocol 1 (a combination of SDS-based solubilization, 1DE-gel concentration and in-gel digestion) showed the best efficiency of coating the amino acid sequence of the external control (Bacillus Megaterium protein BM-3) in the presence of HepaRG extract proteins.