A Comparison of Bottom-Up Proteomic Sample Preparation Methods for the Human Parasite Trichomonas vaginalis

Abstract

Bottom-up proteomic approaches depend on the efficient digestion of proteins into peptides for mass spectrometric analysis. Sample preparation strategies, based on magnetic beads, filter-aided systems, or in-solution digests, are commonly used for proteomic analysis. Time-intensive methods like filter-aided sample preparation (FASP) have led to the development of new, more time-efficient filter-based strategies like suspension trappings (S-Traps) or magnetic bead-based strategies like SP3. S-Traps have been reported as an alternative proteomic sample preparation method as they allow for high sodium dodecyl sulfate (SDS) concentrations to be present in the sample. In this study, we compare the efficiency of different protocols for FASP, SP3, and S-Trap-based digestion of proteins after extraction from Trichomonas vaginalis. Overall, we found a high number of protein IDs for all tested methods and a high degree of reproducibility within each method type. However, FASP with a 3 kDa cutoff filter unit outperformed the other methods analyzed, referring to the number of protein IDs. This is the first work providing the direct comparison of four different bottom-up proteomic approaches regarding the most efficient proteomic sample preparation protocol for the human parasite T. vaginalis.

Figure 1

Protein and peptide identifications of tested sample preparation methods in three biological replicates with two technical replicates combined for each sample preparation method. (A) Overlap of identified protein groups. (B) Overlap of identified peptides. (C) Average number of identified protein groups, peptides, PSMs and MS/MS spectra.

Figure 2

Trypsin efficiency was calculated across all experiments. The percentage of identified peptides containing either zero, one, or two missed cleavages is shown for each sample preparation method. Percentages shown are an average of three biological replicates with two technical replicates each.

Figure 3

Three biological replicates on the protein level. Venn diagrams display an overlap in the identified protein groups as the average of two technical replicates for each digestion condition (FASP 10 kDa, FASP 3 kDa, S-Trap, SP3).

Figure 4

Three biological replicates on the peptide level. Venn diagrams display an overlap in the identified peptides as an average of two technical replicates for each digestion condition (FASP 10 kDa, FASP 3 kDa, S-Trap, SP3).

Figure 5

(A) Hierarchical clustering showing protein expression patterns of T. vaginalis for each sample preparation method. Red bands indicate higher protein expression for one method compared to the others, while the cream-colored bands indicate low protein expression compared to the other methods. (B) PCA score plot of FASP 10 kDa (red), FASP 3 kDa (yellow), S-Trap (blue), and SP3 (green).

Figure 6

Volcano plots displaying the statistical p-value with the magnitude of abundance changes between each sample preparation method compared to FASP of 3 kDa. Red dots indicate proteins meeting the threshold for the significance of changes in protein abundance levels (log₂ < 1 or log₂ > 1 with a p-value of <0.05).