A Modified Orbitrap Tribrid Mass Spectrometer with Real Time Search and Advanced Spectral Processing Enhances Multiplexed Proteome Coverage and Quantification Accuracy

Importance of the Topic

Tandem Mass Tag (TMT)-based quantitative proteomics enables simultaneous analysis of multiple biological samples, advancing our understanding of protein dynamics in health and disease. High plex multiplexing (up to 11 or 16 channels) increases throughput but also introduces co-isolation interference, which can distort quantitative accuracy.

Objectives and Study Overview

This study evaluates a modified Orbitrap Eclipse Tribrid mass spectrometer equipped with real-time search (RTS) capabilities and advanced spectral processing (TurboTMT) to enhance quantitative accuracy, precision, and proteome coverage in TMT SPS MS3 workflows using a standardized yeast digest.

Methodology and Instrumentation

• Sample: Pierce TMT11plex yeast digest standard (four Saccharomyces cerevisiae strains including knockouts).
• Chromatography: EASY-nLC 1200 with EASY-Spray C18 50 cm column; 50 min acetonitrile gradient (8–32% ACN, 0.1% FA) at 45 °C.
• Mass Spectrometry: Modified Orbitrap Eclipse Tribrid with RTS filter, TurboTMT (ΦSDM algorithm), optimized quadrupole, and Ion Routing Multipole.
• Data Processing: Proteome Discoverer 2.3, SEQUEST HT search engine, 10 ppm MS1 tolerance, 0.5 Da MS2 tolerance, 1% FDR, TMT6plex static modification.

Main Results and Discussion

• RTS-triggered MS3 scans occur only upon successful peptide identification, increasing SPS MS3 acquisition efficiency and boosting peptide identifications by ~55% compared to conventional SPS MS3.
• TurboTMT (ΦSDM) resolves TMT reporter ions at 15 000–30 000 resolution in MS2, improving throughput and identification rates without extending transients.
• Hardware modifications, including an optimized quadrupole and improved ion transmission, allow narrower isolation windows (0.7 m/z) and reduce co-isolation interference, achieving ~95% interference-free quantitation.
• Precision improved, with coefficient of variation (CV) reduced to 2–6% across expected TMT ratios and accuracy closely matching theoretical values in complex HeLa spike-in experiments.

Benefits and Practical Applications

The integration of RTS and TurboTMT in the Orbitrap Tribrid platform delivers higher confidence in TMT quantitation, deeper proteome coverage, and faster analytical cycles. This approach is applicable to complex biological studies, clinical biomarker discovery, and high-throughput QA/QC workflows requiring robust multiplexed analyses.

Future Trends and Potential Uses

• Extension to next-generation isobaric tags (16 or more channels) and custom chemical modifications using RTS.
• Integration with ion mobility (FAIMS) and AI-driven acquisition strategies to further reduce interference.
• Real-time adaptive methods for targeted proteomics and single-cell quantitation.

Conclusion

The modified Orbitrap Eclipse Tribrid mass spectrometer with real-time search and TurboTMT enhances the speed, depth, and quantitative accuracy of TMT SPS MS3 workflows, offering a powerful platform for high-plex proteomic investigations.

Reference

1. Paulo et al. A Triple Knockout (TKO) Proteomics Standard for Diagnosing Ion Interference in Isobaric Labeling Experiments. J. Am. Soc. Mass Spectrom. 2016;27:1620.
2. Erickson 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 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.