Discovery with the Orbitrap Eclipse Tribrid mass spectrometer

Significance of the topic

Advanced mass spectrometry instruments are essential for modern analytical chemistry, enabling deeper insights into proteome composition, structural biology and biopharmaceutical characterization. The Orbitrap Eclipse Tribrid mass spectrometer introduces hardware and software innovations that address sensitivity, selectivity and versatility challenges in complex workflows, from single-cell proteomics to native intact protein analysis.

Objectives and overview of the study

• Highlight hardware improvements that enhance ion transmission, mass range and vacuum performance
• Describe software advances, including Real-Time Search and prebuilt method templates
• Demonstrate benefits in quantitative proteomics with TMT multiplexing and single-cell sensitivity
• Illustrate capabilities in intact protein and native complex analysis

Methodology and instrumentation

Key innovations and configurations include

• QR5 segmented quadrupole mass filter with hyperbolic surfaces for improved sensitivity at narrow isolation widths
• Ultra-high-field Orbitrap analyzer delivering up to 40 Hz MS2 rates at high resolution
• Dual-pressure linear ion trap with extended high-pressure cell for multiple fragmentation modes (CID, HCD, ETD, ETciD, EThcD and UVPD)
• Enhanced vacuum manifold in the C-trap and Orbitrap regions for intact protein analysis
• FAIMS Pro interface for gas-phase fractionation and orthogonal selectivity
• Real-Time Search using Comet engine for on-the-fly peptide identification in SPS MS3 workflows
• Optional PTCR source for proton transfer charge reduction in top-down and native MS
• Instrument Control Software with prebuilt and customizable acquisition templates

Main results and discussion

Real-Time Search integrated into SPS MS3 workflows increased quantified peptide identifications by up to 38 percent and protein quantitation accuracy compared to classic SPS MS3, while approaching MS2 depth. FAIMS Pro interface reduced singly charged interference, boosting proteome coverage by over 50 percent in label-free single-cell analyses. Combining FAIMS with linear trap MS2 detection and HCD fragmentation yielded over 8500 peptides and 7300 proteins from 0.5 ng HeLa digest. Native MS with high-mass precursor isolation and PTCR enabled deconvolution of complex Cytokine-Fc fusion proteoforms. Stepped FAIMS compensation voltages delivered rapid separation of mAb light and heavy chains for accelerated subunit characterization.

Benefits and practical applications

• High-throughput multiplexed quantitation with enhanced TMT accuracy and dynamic range
• Superior single-cell proteome coverage for cell-type profiling and translational research
• Comprehensive top-down and native analysis of intact proteins and complexes
• Automated prebuilt workflows reduce method development time across disciplines

Future trends and potential applications

Continued advances in real-time data acquisition and AI-driven decision making will drive deeper proteome and interactome mapping. Expanded multiplexing beyond 16plex and integration with microfluidic sample preparation will further increase throughput. Combining multi-omics data in real time and advanced fragmentation schemes promises new frontiers in single-cell and structural proteomics.

Conclusion

The Orbitrap Eclipse Tribrid mass spectrometer represents a leap forward in sensitivity, selectivity and flexibility. Its suite of hardware and software innovations empowers researchers to tackle the most demanding applications, from single-cell discovery to in-depth native protein characterization.

Reference

• McAlister GC et al Analytical Chemistry 2014 86(14) 7150-7158 DOI 10.1021/ac502040v
• Schweppe DK et al Journal of Proteome Research 2020 19(5) 2026-2034
• Robitaille AM et al Application Note 65729 Thermo Fisher Scientific 2021
• Cong Y et al Analytical Chemistry 2020 92(3) 2665-2671
• Motamedchaboki K Lopez-Ferrer D White Paper 65730 Thermo Fisher Scientific
• Motamedchaboki K et al Application Poster AP-65714 Thermo Fisher Scientific
• Huguet R et al Analytical Chemistry 2019 91(24) 15732-15739
• Melani RD et al mAbs 2019 11(8) 1351-1357
• Thermo Fisher Scientific White Paper 65653 Biopharmaceutical Characterization