Extending Capabilities of Real-Time Database Search on the Orbitrap Eclipse Tribrid Mass Spectrometer for Multiplexed Proteomics

Significance of the topic

Multiplexed proteomics relies on simultaneous measurement of many samples using isobaric tags. Real time database search on high performance Orbitrap instruments addresses the need for efficient, accurate and high‐throughput quantitation. This capability directly impacts exploratory and targeted protein assays in academic and industrial laboratories by improving data quality and reducing instrument time.

Objectives and Overview

This study evaluates enhancements to the Real‐Time Search MS3 workflow for Tandem Mass Tag quantitation on the Orbitrap Eclipse Tribrid mass spectrometer. Key goals include assessing a Close‐Out function that stops further MS3 events once sufficient quantitative data are acquired per protein and implementing real‐time false discovery rate filtering to trigger MS3 scans only for high‐confidence peptide spectrum matches.

Materials and Methods

Samples comprised a Thermo Scientific Pierce TMT 11‐plex yeast digest and TMTpro 16‐plex mixtures of HeLa and E coli peptides spiked at defined ratios. Chromatographic separation used either a Thermo Scientific Easy‐nLC 1000 or an Ultimate 3000 RSLC system with 50 cm EASY‐Spray columns and extended gradients. Both experiments ran on the Orbitrap Eclipse Tribrid mass spectrometer under the instrument control software build ICSW 3.4. The Comet algorithm performed online MS2 database searches within the Real‐Time Search framework. Post‐acquisition analysis employed Sequest HT in Proteome Discoverer 2.5.

Used Instrumentation

• Orbitrap Eclipse Tribrid mass spectrometer
• Thermo Scientific Easy‐nLC 1000 and Ultimate 3000 RSLC systems
• 50 cm EASY‐Spray columns
• Thermo Scientific Pierce TMT11plex Yeast Digest Standard
• TMTpro 16plex labeling reagents

Main Results and Discussion

Integration of Real‐Time Search with SPS‐MS3 increased the number of quantified proteins by 17 and 13 percent over standard SPS‐MS3 workflows, respectively. The Close‐Out feature further boosted protein IDs by up to 26 percent by halting redundant MS3 events once quantitative depth was reached. Real‐time FDR filtering enhanced peptide and spectrum counts by 14 percent. Quantitative precision remained high, with over 95 percent of peptides showing coefficients of variation below 20 percent at reporter ion signal‐to‐noise ratios above ten. RTS informed selection of TMT‐containing fragments reduced interference compared to abundance‐driven isolation.

Benefits and Practical Applications

• Higher throughput through dynamic adjustment of MS3 events
• Improved quantitative accuracy and data completeness
• Reduced instrument time per sample and better resource utilization
• Applicability to complex proteome studies, biomarker discovery and QC workflows

Future Trends and Opportunities

Further developments may integrate machine learning to refine real‐time decision making and expand compatibility with additional labeling strategies. Combining RTS with ion mobility and parallel reaction monitoring could open new frontiers in targeted quantitation and single‐cell proteomics.

Conclusion

Enhanced Real‐Time Search capabilities on the Orbitrap Eclipse deliver significant gains in multiplexed proteomic quantitation. Close‐Out and FDR filtering features streamline MS3 acquisition, maximize identification rates and maintain high precision, offering robust workflows for diverse research and quality control applications.

References

Liu Y Barshop WD Hakimi A Canterbury JD Huguet R Viner R Extending Capabilities of Real‐Time Database Search on the Orbitrap Eclipse Tribrid Mass Spectrometer for Multiplexed Proteomics Thermo Fisher Scientific 2020