An Orbitrap Eclipse Tribrid Mass Spectrometer with Real-Time Search Enhances Multiplexed Proteome Coverage and Quantitation Accuracy

Significance of the Topic

Multiplexed quantitative proteomics using Tandem Mass Tags (TMT) has become a cornerstone for investigating protein abundance and interactions across multiple biological samples in a single experiment. Addressing issues of co-isolation interference and achieving high depth of coverage are critical for accurate and reproducible quantification in complex proteomes.

Objectives and Study Overview

This work evaluates the performance gains of the Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer equipped with Real-Time Search (RTS) capabilities, advanced spectral processing algorithms (TurboTMT driven by ΦSDM), and a redesigned quadrupole. The study benchmarks improvements in identification rates, quantitation accuracy, and proteome coverage for TMT11plex experiments.

Methodology

• Sample: Pierce TMT11plex Yeast Digest Standard (four Saccharomyces cerevisiae strains labeled across 11 channels).
• Chromatography: 50 min gradient (8–32% acetonitrile, 0.1% formic acid) on an EASY-nLC 1200 with a 50 cm EASY-Spray C18 column at 45 °C.
• Acquisition: Orbitrap Eclipse Tribrid mass spectrometer. Three methods compared: conventional MS², SPS MS³, and RTS-triggered SPS MS³. MS1 scans at 120 000 resolution; MS2/MS3 conditions optimized per strategy; dynamic exclusion 45 s.
• Data Processing: Proteome Discoverer 2.3 with SEQUEST HT, 10 ppm MS1 and 0.5 Da MS2 tolerances, 1% FDR, static TMT6plex modification.

Used Instrumentation

• Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer (modified dual-pressure linear ion trap, ultra-high-field Orbitrap analyzer, segmented quadrupole).
• Thermo Scientific EASY-nLC 1200 UHPLC system with EASY-Spray ion source.
• ΦSDM algorithm (TurboTMT) for enhanced resolution of TMT reporter ions.

Key Results and Discussion

• Real-Time Search reduced false triggers by initiating MS³ scans only upon confident peptide-spectrum matches (PSMs), increasing valid SPS MS³ events by ~55% compared to conventional SPS.
• TurboTMT (ΦSDM) improved reporter-ion resolution at lower transient lengths (15 000–30 000 resolving power), accelerating MS2 acquisition rate and boosting peptide identifications.
• Optimized quadrupole transmission allowed narrower isolation windows, further reducing co-isolation interference and enhancing quantitative accuracy (>95% interference-free measurements).
• Precision (CV) improved from ~10–15% in standard SPS MS³ to ~2–4% using RTS SPS MS³, and accuracy of measured ratios closely matched expected values across a dynamic range.

Benefits and Practical Applications

• Enhanced multiplexing throughput and confidence in quantitative proteomics workflows for biological, pharmaceutical, and clinical research.
• Reduction of analytical overhead by minimizing unproductive scans, maximizing data acquisition efficiency.
• Applicability to diverse sample types and increased compatibility with high-plex isobaric tagging strategies.

Future Trends and Potential Applications

• Integration of machine-learning algorithms for real-time decision-making and dynamic method adaptation.
• Expansion to next-generation isobaric tags with higher plexing capacity and custom modification workflows.
• Synergy with ion mobility and additional gas-phase separation techniques to further refine selectivity and depth.

Conclusion

The modified Orbitrap Eclipse Tribrid platform with Real-Time Search and TurboTMT processing delivers significant gains in proteome coverage, quantitative precision, and accuracy for multiplexed TMT experiments. These advances streamline high-throughput proteomic analyses and set the stage for further innovations in real-time mass spectrometric workflows.

References

1. Paulo J. A. et al. A Triple Knockout (TKO) Proteomics Standard for Diagnosing Ion Interference in Isobaric Labeling Experiments. J. Am. Soc. Mass Spectrom. 2016;27(10):1620–1628.
2. Erickson B. K. et al. Active Instrument Engagement Combined with a Real-Time Database Search for Improved Performance of Sample Multiplexing Workflows. J. Proteome Res. 2019;18(3):1299–1306.
3. Schweppe D. K. et al. Characterization and Optimization of Multiplexed Quantitative Analyses Using High-Field Asymmetric-Waveform Ion Mobility Mass Spectrometry. Anal. Chem. 2019;91(6):4010–4016.