Rapid Identification of Bacillus Spores by MALDImini-1 Compact MALDI Digital Ion Trap (DIT) Mass Spectrometer

Importance of the Topic

Plate-based proteolytic profiling using MALDI-TOF MS has become essential for rapid and accurate identification of microbial species. Conventional MALDI-TOF workflows struggle to analyze spore-forming bacteria and complex mixtures, presenting challenges in diagnostics, food safety, and biodefense. Developing accelerated methods for spore detection addresses critical needs in clinical microbiology and biosecurity.

Goals and Study Overview

We aimed to devise a streamlined approach for identifying Bacillus spores using on-target enzymatic digestion followed by MALDI-MS/MS. The study focused on Bacillus subtilis spores as a model, evaluating a compact MALDI-DIT instrument for rapid proteomic analysis.

Methodology and Instrumentation

Sample Preparation Workflow:

• Spore formation: Cultivation of Bacillus subtilis subsp. subtilis NBRC 13719T on nutrient agar at 30 °C for a minimum of one week; confirmation via microscopy.
• Cell lysis and protein extraction: Application of bacterial smear on MALDI target; addition of 10% trifluoroacetic acid; drying.
• Immobilized trypsin digestion: Addition of trypsin beads; incubation at 37 °C for 30 minutes under humid conditions.
• Matrix application: Overlay of α-cyano-4-hydroxycinnamic acid solution; drying.
• Mass spectrometry: Acquisition of MS and MS/MS spectra using the MALDImini-1 compact MALDI-DIT.

Instrumentation Used:

• MALDImini-1 compact MALDI-DIT mass spectrometer
• Trypsin-immobilized beads (ProteoChem)
• α-Cyano-4-hydroxycinnamic acid matrix solution
• 10% trifluoroacetic acid

Main Results and Discussion

• MS spectra of tryptic digests revealed multiple peptide peaks; m/z 1879 was selected for MS/MS fragmentation.
• Database search (Mascot) identified the peptide as a small acid-soluble spore protein (SASP) from B. subtilis.
• BLAST verification confirmed sequence uniqueness to B. subtilis, distinguishing it from B. anthracis and B. cereus.
• These findings validate the feasibility of rapid on-plate digestion coupled with MALDI-MS/MS for species-level spore identification.

Benefits and Practical Applications

• Substantially reduced digestion time (30 minutes) compared to conventional overnight protocols.
• Compact instrument design enables near-sample analysis, enhancing workflow efficiency in constrained laboratory spaces.
• Applicable to rapid pathogen identification in clinical, food safety, and biodefense scenarios.

Future Trends and Potential Applications

• Extension of workflow to mixed microbial communities and other spore-forming genera.
• Integration with automated sample preparation platforms for higher throughput.
• Development of robust reference libraries for comprehensive MS/MS-based microbial identification.

Conclusion

The presented approach combines on-plate proteolysis with compact MALDI-DIT MS/MS to rapidly and specifically identify Bacillus spores. The use of immobilized trypsin and a bench-top DIT system achieves fast turnaround, offering a versatile platform for microbial diagnostics in limited-space environments.

References

• B. Warscheid and C. Fenselau (2003) Characterization of Bacillus Spore Species and Their Mixtures Using Postsource Decay with a Curved-Field Reflectron. Analytical Chemistry 75:5618–5627.