Comparison of In-Solution, FASP, and S‑Trap Based Digestion Methods for Bottom-Up Proteomic Studies

Bottom-up proteomic
strategies rely on efficient digestion of proteins
into peptides for mass spectrometry analysis. In-solution and filter-based
strategies are commonly used for proteomic analysis. In recent years,
filter-aided sample preparation (FASP) has become the dominant filter-based
method due to its ability to remove SDS prior to mass spectrometry
analysis. However, the time-consuming nature of FASP protocols have
led to the development of new filter-based strategies. Suspension
traps (S-Traps) were recently reported as an alternative to FASP and
in-solution strategies as they allow for high concentrations of SDS
in a fraction of the time of a typical FASP protocol. In this study,
we compare the yields from in-solution, FASP, and S-Trap based digestions
of proteins extracted in SDS and urea-based lysis buffers. We performed
label-free quantification to analyze the differences in the portions
of the proteome identified using each method. Overall, our results
show that each digestion method had a high degree of reproducibility
within the method type. However, S-Traps outperformed FASP and in-solution
digestions by providing the most efficient digestion with the greatest
number of unique protein identifications. This is the first work to
provide a direct quantitative comparison of two filter-based digestion
methods and a traditional in-solution approach to provide information
regarding the most efficient proteomic preparation.