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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.

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