Developments in Real-Time Search on an Orbitrap Tribrid Mass Spectrometer

Importance of the Topic

Real time database searching during LC MS workflows allows on the fly decisions that enhance proteome coverage and instrument efficiency. Integrating rapid search results with dynamic fragmentation selection supports deeper analysis and better use of MS instrument time.

Objectives and Study Overview

This study aimed to expand the Real Time Search feature on Orbitrap Tribrid mass spectrometers by introducing:

• Multi feature filtering using a real time estimate of false discovery rate (FDR).
• Protein "close out" to limit repeated identification of the same proteins beyond a user defined count.
• Support for precursor and fragment neutral loss in the Comet search engine.

These capabilities were tested on TMT labeled yeast peptides to assess improvements in sensitivity and throughput.

Methodology and Instrumentation

The workflow used a Thermo Scientific Easy nLC 1200 system with a two hour gradient on EASY Spray columns coupled to an Orbitrap Eclipse Tribrid MS. A Comet based search engine was embedded in the instrument control software to perform MS2 spectral matching in real time on a host PC. Features such as Xcorr, deltaCn, mass accuracy, ion matching metrics, peptide length, and charge state were combined via linear discriminant analysis. Target and decoy PSMs were accumulated, and every 1000 target PSMs the discriminant model was retrained to maintain a 20 percent FDR threshold.

Main Findings and Discussion

• Real time FDR filtering using multi feature scoring improved confidence in PSM selection without impacting instrument cycle time.
• Close out filtering reduced repeated analysis of abundant proteins and increased MS3 trigger events by approximately 10 percent in triplicate runs.
• Neutral loss support in the Comet engine enabled additional identification of modified peptides though data were not shown in this poster.

Dynamic score threshold updates and multithreaded processing ensured seamless integration of search and acquisition processes.

Benefits and Practical Applications

The enhanced RTS approach maximizes use of MS instrument time by focusing acquisition on novel peptide identifications. It is particularly valuable for complex or fractionated samples where excluding common proteins can improve depth of coverage.

Future Trends and Potential Applications

• Real time localization of post translational modifications to guide targeted fragmentation.
• Application to multi fraction and single cell proteomics for greater discrimination of low abundance targets.
• Integration of advanced machine learning models for adaptive scoring and dynamic method updates.

Conclusion

Implementing FDR based filtering, protein close out, and neutral loss support in real time enhances the sensitivity and throughput of Orbitrap Tribrid analyses. These developments pave the way for more intelligent acquisition strategies in proteomics.

References

• Schweppe et al J Proteome Res 2020 19 2026–2034
• Erickson et al J Proteome Res 2019 18 1299–1306
• Eng et al JASMS 2015 26 1865
• Accord NET Framework v 3.8.0