Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer

Importance of the Topic

Mass spectrometry is essential in analytical chemistry for resolving complex molecular structures, quantifying low-abundance analytes, and driving discoveries in proteomics, structural biology, and biopharmaceutical characterization.

The ability to distinguish subtle differences in peptides, proteins, and complexes underpins research in disease mechanisms, biomarker discovery, and drug development.

Tribrid mass spectrometers with advanced ion manipulation and scanning capabilities address limitations of sensitivity, selectivity, and throughput in current workflows.

Objectives and Overview of the Article

This article presents the Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer, detailing its novel design features, enhanced performance, and application examples.

Key aims include demonstrating improvements in single-cell proteomics, high-throughput quantitative workflows, native protein and protein–ligand analysis, and top-down proteoform characterization.

Methodology

The instrument builds upon the Tribrid architecture by integrating advanced ion transmission, extended m/z range, and real-time decision algorithms.

Novel techniques include Real-Time Search for on-the-fly peptide identification, Proton Transfer Charge Reduction (PTCR) for charge state manipulation, High Mass Range MSn (HMRn) for intact complex analysis, and flexible fragmentation modes (CID, HCD, ETD, EThcD, UVPD).

Examples of workflows:

• MS3-based Tandem Mass Tag (TMT) labeling with Real-Time Search for single-cell proteomics.
• TMTpro 16plex quantitation with boosted throughput and accuracy via on-the-fly MS3 selection.
• Native MS of therapeutic proteins using HMRn and PTCR to resolve glycoforms and noncovalent assemblies.
• Top-down proteoform analysis facilitated by PTCR to reduce spectral complexity.
• Membrane protein–ligand identification employing pseudo-MS2 and MSn steps to isolate and fragment bound ligands.

Used Instrumentation

Orbitrap Eclipse Tribrid mass spectrometer equipped with:

• Quadrupole mass filter with segmented hyperbolic surfaces
• Advanced active ion beam guide and EASY-IC calibration source
• Electrodynamic ion funnel and high-capacity transfer tube
• Dual-pressure linear ion trap for MSn with extended fragmentation options
• FAIMS Pro interface, EASY-ETD, PTCR, HMRn, and UVPD modules

Key Results and Discussion

Single-cell proteomics achieved quantitation of hundreds of differentially expressed proteins per cell using MS3 TMT workflows with Real-Time Search and TMTpro reagents.

TMTpro 16plex with Real-Time Search improved peptide coverage by over 38% and protein quantification depth by 53% compared to standard SPS MS3, halving analysis time.

Native MS of an Fc-fusion cytokine and therapeutic antibodies revealed over 30 distinct glycoforms and detailed complementarity determining region (CDR3) sequences using combined fragmentation maps.

Protein–ligand complexes were analyzed natively, with top-down MSn enabling unambiguous identification of small molecules bound to membrane proteins.

PTCR enhanced top-down data acquisition by expanding identifiable proteoforms from overlapping charge state distributions, increasing the number of characterized species.

Benefits and Practical Applications

Enhanced sensitivity and selectivity enable ultra-low sample analysis, including single cells and intact protein complexes.

Real-Time Search optimizes instrument duty cycle, delivering high-throughput, accurate quantitation across up to 16 multiplexed samples in a single run.

Comprehensive native and top-down workflows facilitate characterization of biotherapeutics, membrane assemblies, and complex proteoform mixtures.

Future Trends and Possibilities

Integration of real-time data analytics and AI-driven acquisition strategies to further boost throughput and discovery potential.

Expansion of multiplexed single-cell analyses to deeper proteome coverage and integration with spatial omics.

Advances in ion mobility and photodissociation techniques for enhanced structural resolution of large biomolecular assemblies.

Broader adoption of native top-down MSn for routine drug–target interaction studies and quality control in biopharmaceutical pipelines.

Conclusion

The Orbitrap Eclipse Tribrid mass spectrometer represents a significant advancement in analytical capabilities across proteomics, structural biology, and biopharmaceutical characterization.

Its combination of ion manipulation, real-time decision making, and versatile fragmentation modes delivers unprecedented sensitivity, throughput, and depth of analysis.

These innovations pave the way for new discoveries in single-cell biology, top-down proteomics, and native complex characterization.

References

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4. Gault, J. et al. Nature Methods, in press.
5. Huguet, R. et al. Analytical Chemistry, 2019, 91(24).