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.