Small Molecule Real-Time Library Search

Significance of the Topic

Real-Time Library Search for small molecules represents a significant advancement in mass spectrometry, enabling on-the-fly comparison of acquired MS2 spectra with reference libraries. This capability enhances data quality and decision making by directing subsequent fragmentation steps toward the most informative ions.

Objectives and Study Overview

The study aimed to integrate spectral library searching into the Orbitrap IQ-X and Eclipse Tribrid platforms via the Tune 3.5 control software. Key goals included:

• Implementing real-time comparison of experimental spectra against mzVault and mzCloud libraries.
• Utilizing similarity scores to guide acquisition logic (e.g., MS3 triggering, collision energy adjustments).
• Maintaining rapid processing to avoid delaying instrument throughput.

Methodology and Instrumentation Used

The Real-Time Library Search feature extends the existing Real-Time Search infrastructure in Tune 3.5. MS2 scans (ddMS2) are streamed to a backend service (Glutamate), which filters candidate library entries by polarity, activation mode, collision energy tolerance, adduct offsets, and ppm mass tolerance. Cosine similarity scores and confidence metrics are computed for each candidate. The highest matches inform dynamic exclusion lists or trigger further MSn scans. Instruments and software:

• Thermo Scientific Orbitrap IQ-X Tribrid MS
• Thermo Scientific Orbitrap Eclipse Tribrid MS
• Tune Instrument Control Software 3.5
• mzVault and mzCloud (.db format) spectral libraries

Key Results and Discussion

Real-Time Library Search successfully generated cosine similarity and confidence scores during acquisition without extending cycle times. The system could discriminate high-confidence matches, suggest optimized collision energies for under-fragmented ions, and refine precursor selection based on library hits. Method templates demonstrated flexible experimental workflows, from simple identification to nested MSn branching.

Benefits and Practical Applications

By embedding spectral library comparisons directly into the acquisition process, users can:

• Improve specificity of molecular identifications in complex matrices.
• Optimize collision energies dynamically to enhance fragmentation quality.
• Automate decision logic for MSn acquisitions, reducing method development time.

Future Trends and Opportunities

Advances may include integration with machine-learned fragmentation prediction, expanded cloud library access, and support for even larger adduct and isotope variants. Real-time adaptation to metabolomics, lipidomics, and environmental analyses will further extend the utility of this approach.

Conclusion

Real-Time Library Search on Orbitrap Tribrid platforms combines fast spectral matching with intelligent acquisition logic, improving data quality and experimental efficiency. The method’s compatibility with custom and public libraries positions it as a valuable tool for diverse small molecule applications.

References

• Barshop W, Canterbury J, Bills B, Sharma S. Small Molecule Real-Time Library Search. Thermo Fisher Scientific.