Identification of Small Molecules via Real-Time Library Search on an Orbitrap Tribrid Mass Spectrometer

Importance of the Topic

Real-time spectral library searching integrated with data acquisition enables adaptive workflows that improve identification and quantitation of small molecules in complex samples. This capability supports dynamic method adjustments and enhances the depth of small-molecule profiling in pharmaceutical, environmental, and metabolomics applications.

Study Objectives and Overview

This study aimed to extend real-time search technology, originally developed for peptide analysis, to small molecule identification on Thermo Fisher Scientific Orbitrap Eclipse and IQ-X Tribrid mass spectrometers. The authors developed version 3.5 of the Tune instrument control software to perform a concurrent MS2 spectral library search (Real-Time Library Search, RTLS) and to use search outcomes to drive subsequent MSn acquisition decisions.

Methodology and Instrumentation

The RTLS workflow is implemented as a Windows service (C#) running alongside the Tune control software. Key methodological features include:

• Spectral Library Input: mzVault .db format libraries, including offline copies of the mzCloud MS2 database.
• Search Parameters: collision energy tolerances, precursor mass tolerance, permissible adduct forms, and maximum per-scan search duration.
• Scoring Metrics: cosine similarity and confidence scores used to trigger downstream scans based on user-defined thresholds.
• Decision Logic: multiple filter instances can be configured to apply complex acquisition strategies such as targeted MSn, background exclusion, or automatic reinjection.

Instrumentation details:

• Instrument Control Software: Thermo Fisher Tune version 3.5.
• Mass Analyzers: Orbitrap Eclipse and IQ-X Tribrid mass spectrometers operating in Small Molecule Application Mode.
• Compute Platform: Embedded Windows service communicating with instrument via a companion PC.

Main Results and Discussion

Implementation of RTLS on both the IQ-X and Eclipse platforms allowed sub–100 ms library searches per MS2 scan (median durations derived from 15,130 scans), enabling on-the-fly identification without compromising duty cycle. The approach successfully:

• Filtered MS2 events in real time to select precursors for targeted MS3 or alternative activation modes.
• Excluded background ions and previously fragmented precursors to focus on analytes of interest.
• Supported multi-instance filters for parallel acquisition logic and complex decision trees.

Search performance remained robust across a wide range of collision energies (5–65 NCE) and within ±5 ppm mass tolerances. Scalability to large spectral libraries (over 2 M spectra in mzCloud) was demonstrated through efficient in-memory indexing and adduct offset handling.

Benefits and Practical Applications

• Enhanced Specificity: Real-time recognition of target compounds guides subsequent MSn acquisitions, increasing confidence in identification.
• Improved Throughput: Dynamic exclusion and reinjection schemes maximize sampling of low-abundance species.
• Flexible Method Design: User-defined score thresholds and compound-class filters enable customized acquisition strategies for QA/QC, metabolite profiling, and environmental monitoring.

Future Trends and Applications

• Integration with Machine Learning: Adaptive scoring models to refine real-time identification accuracy.
• Cloud-Enabled Libraries: Streaming and updating spectral databases in real time for broader compound coverage.
• Expanded Untargeted Workflows: Coupling RTLS with ion mobility or data-independent acquisition for comprehensive metabolomics.
• Automated Annotation Pipelines: Linking real-time MS data to cheminformatics platforms and pathway analysis tools.

Conclusion

Real-Time Library Search on Orbitrap Tribrid platforms represents a significant advance in small molecule mass spectrometry, uniting high-resolution instrumentation with agile data-driven acquisition. By leveraging spectral libraries during run time, RTLS enhances selectivity, throughput, and methodological flexibility across diverse analytical applications.

References

• Erickson BK et al. (2019) Active Instrument Engagement Combined with a Real-Time Database Search for Improved Performance of Sample Multiplexing Workflows. J Proteome Res 18(3):1299–1306.
• Eng JK et al. (2013) Comet: an open-source MS/MS sequence database search tool. Proteomics 13(1):22–24.
• Thermo Fisher Scientific (2020) mzVault .db and mzCloud Spectral Libraries User Guide.