In-Depth Comparison of Reagent-Based Digestion Methods and Two Commercially Available Kits for Bottom-Up Proteomics

Proteomic analysis plays an essential role in biology with several methodologies available for sample preparation and analysis. This study evaluates and compares various cell lysis and protein digestion protocols for bottom-up proteomics using HeLa S3 cells. We assessed two physical disruption methods to homogenize cells-sonication and BeatBox-alongside four digestion protocols. Two of them are lab-reagent strategies: urea-based and sodium deoxycholate (SDC)-based in-solution digestion, and two are commercially available kits: the EasyPep kit from Thermo Fisher Scientific and S-Trap from Protifi. Each method's efficacy was evaluated based on protein recovery, peptide yield, and number of unique proteins identified through LC-MS analysis. Our results indicate that while both sonication and the BeatBox (PreOmics Inc.) methods provided comparable protein recovery and coverage, the choice of digestion method had a much bigger impact on the amount of protein IDs found. SDC digestion yielded the highest protein and peptide counts, while S-Trap exhibited the most consistent peptide recovery. Conversely, EasyPep showed higher variability in peptide recovery, with a ±10% difference in the average peptide number. Each homogenization strategy and digestion method also yielded its own list of unique proteins. These results provide several lists of proteins for biologists to select from based on experimental needs and highlight the importance of choosing appropriate protocols for comprehensive proteomic analyses.