Mass Spectrometry-based Deep Coverage Proteome: Evaluation of Cellular Protein Extraction Methods

The current study comprehensively evaluates four different protein extraction methods based on urea, sodium dodecyl sulfate(SDS), anionic surfactants(BT), and total RNA extractor(Trizol), aiming to optimize the sample preparation workflow for mass spectrometry-based proteomics. Using HeLa cells as an example, we found that the method employing the mass spectrometry-compatible surfactant BT reagent significantly reduces the total time consumed for protein extraction and minimizes protein losses during the sample preparation process. Further integrating the four protein extraction methods, we identified over 7000 proteins from HeLa cells without relying on pre-fractionation techniques, and 2990 of them were quantified using label-free quantification. It is worth noting that the BT and SDS methods demonstrate higher efficiency in extracting membrane proteins, while the Urea and Trizol methods are more effective in extracting proteins from nuclear and cytoplasmic fractions. In summary, this study provides a novel solution for deep proteome coverage, particularly in the context of cellular protein extraction, by integrating mass spectrometry-compatible surfactants with traditional extraction methods to effectively enhance protein identification numbers.