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

Abstract

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.

Improving Proteome Coverage for Small Sample Amounts: An Advanced Method for Proteomics Approaches with Low Bacterial Cell Numbers

“…In the last years, different sample preparation methods have emerged and been adapted. [15] However, each additional processing step, e.g., removal of interfering compounds, results in sample loss, particularly when starting with low cell numbers, severely compromising the number of proteins covered. The single pot solid-phase enhanced sample preparation protocol (SP3 protocol) uses magnetic beads for the sample preparation and was first introduced by Hughes et al [16] This protocol displays increased efficiency, speed, throughput, and robustness to, e.g., solvents, pH, or sample complexity for sample processing in proteomics of eukaryotic cells.…”

“…However, not only enrichment of bacteria is of importance, but also optimized cell lysis and sample preparation for LC–MS/MS analysis. In the last years, different sample preparation methods have emerged and been adapted . However, each additional processing step, e.g., removal of interfering compounds, results in sample loss, particularly when starting with low cell numbers, severely compromising the number of proteins covered.…”

Characterization of a Human Liver NIST Reference Material fit for Proteomics Applications

“…The development and availability of a common material for quality control purposes would significantly improve the critical evaluation of sample comparisons and processing and the variability of results. [5][6][7] We report a data set of peptide identifications from RM 8461 Human Liver for Proteomics, a cryogenically homogenized and freeze-dried liver tissue developed as a material for complex proteomic analysis. The data set was collected using high resolution LC/MS instrumentation typically utilized in top-down and bottom-up protein analysis.…”

Comparison of protein and peptide fractionation approaches in protein identification and quantification fromSaccharomyces cerevisiae

“…FASP has become a widely used method for in-solution digestion of proteins due to its ability to remove detergents prior to mass spectrometry analysis [5]. The FASP method uses a common ultrafiltration device whereby the membrane pores are small enough to allow contaminating detergents to pass through, while proteins are retained and concentrated in the filter unit [24].…”