Evaluation of Sample Preparation Methods for Fast Proteotyping of Microorganisms by Tandem Mass Spectrometry

Abstract

Tandem mass spectrometry-based proteotyping allows characterizing microorganisms in terms of taxonomy and is becoming an important tool for investigating microbial diversity from several ecosystems. Fast and automatable sample preparation for obtaining peptide pools amenable to tandem mass spectrometry is necessary for enabling proteotyping as a high-throughput method. First, the protocol to increase the yield of lysis of several representative bacterial and eukaryotic microorganisms was optimized by using a long and drastic bead-beating setting with 0.1 mm silica beads, 0.1 and 0.5 mm glass beads, in presence of detergents. Then, three different methods to obtain greater digestion yield from these extracts were tested and optimized for improve efficiency and reduce application time: denaturing electrophoresis of proteins and in-gel proteolysis, suspension-trapping filter-based approach (S-Trap) and, solid-phase-enhanced sample preparation named SP3. The latter method outperforms the other two in terms of speed and delivers also more peptides and proteins than with the in-gel proteolysis (2.2 fold for both) and S-trap approaches (1.3 and 1.2 fold, respectively). Thus, SP3 directly improves tandem mass spectrometry proteotyping.

Keywords: detection; identification; mass spectrometry; microorganisms; proteotyping; sample preparation; shotgun proteomics.

FIGURE 1

Overview of protein extraction and proteolysis comparison methods for a fast and performant sample preparation approach for proteotyping.

FIGURE 2

Protein extraction efficiency by bead beating for B. subtilis and S. cerevisiae samples compared to E. coli. Proteins obtained by bead beating (10 cycles of 30 s at 10,000 rpm) were subjected to denaturing electrophoresis and after Coomassie staining, samples were compared by densitometry (n = 2). The red line represents the E. coli densitometric reference, blue bar chart represents the B. subtilis densitometry ratio while gray one represents the S. cerevisiae densitometry ratio. Silica, silica beads 0.1 mm; Glass_A, glass beads 0.5 mm; Glass_B, glass beads 0.1 mm; BMA (Beads Mixture A), 2/3 silica beads 0.1 mm + 1/3 glass beads 0.5 mm; BMB (Beads Mixture B), 2/3 silica beads 0.1 mm + 1/3 glass beads 0.1 mm; BMC (Beads Mixture C), 1/3 silica beads 0.1 mm + 1/3 glass beads 0.5 mm + 1/3 glass beads 0.1 mm. Densitometry measurements are detailed on Supplementary Table S1.

FIGURE 3

Proteomics-based comparison of mixtures of beads for cell lysis of Mix3. (A) NanoLC-MS/MS data for the Mix3 sample that includes B. subtilis (55%), E. coli (35%), and S. cerevisiae (10%) lysed either with BMA or BMC (n = 3). Only proteins validated with at least two peptides are considered (Supplementary Table S2). (B) Peptide and protein distributions amongst the three organisms present in the sample: B. subtilis (orange), E. coli (yellow) and S. cerevisiae (green).

FIGURE 4

Comparison of 60 min and 15 min in-gel proteolysis performances for Mix3. (A) NanoLC-MS/MS data for the triplicate analysis. Only proteins validated with at least two peptides are considered (Supplementary Table S2). (B) Peptide and protein distribution amongst the three organisms present in the sample: B. subtilis (orange), E. coli (yellow), and S. cerevisiae (green). (C) Proportion of peptides with 0 (green bars), 1 (blue bars) or 2 (yellow bars) missed cleavages. (D) Distribution of isoelectric point and hydrophobicity of peptides. The ratio of peptides obtained after 60 min and 15 min proteolysis are represented with blue and orange bars, respectively.

FIGURE 5

Comparison of 60 min and 15 min in-solution proteolysis of Mix3 using S-Trap protein purification mini-columns. (A) NanoLC-MS/MS data for the triplicate analysis. (B) Peptide and protein distribution amongst the three organisms present in the sample: B. subtilis (orange), E. coli (yellow) and S. cerevisiae (green). (C) Proportion of peptides with 0 (green bars), 1 (blue bars) or 2 (yellow bars) missed cleavages. (D) Distribution of isoelectric point and hydrophobicity of peptides. The ratio of peptides obtained after 60 min and 15 min proteolysis are represented with gray and yellow bars, respectively.

FIGURE 6

Comparison of 60 min and 15 min SP3 in-solution proteolysis of Mix3. (A) NanoLC-MS/MS data for the triplicate analysis. (B) Peptide and protein distribution amongst the three organisms present in the sample: B. subtilis (orange), E. coli (yellow), and S. cerevisiae (green). (C) Proportion of peptides with 0 (green bars), 1 (blue bars) or 2 (yellow bars) missed cleavages. (D) Distribution of isoelectric point and hydrophobicity of peptides. The ratio of peptides obtained after 60 min and 15 min proteolysis are represented with brown and green bars, respectively.

FIGURE 7

Comparison of in-gel (Gel_15 min), S-Trap (S-Trap_15 min) and SP3 (SP3_15 min) optimized protocols. (A) Venn diagram showing the overlap of peptide sequences identified for each sample preparation methods. (B) Venn diagram showing the overlap of proteins validated with at least two different peptides for each sample preparation methods. (C) Schematic representation of the workflow timing for the three sample preparation methods (numbers indicated the time in min required per step).