Grant application resource - Orbitrap Ascend Tribrid mass spectrometer

Significance of the topic

Mass spectrometry continues to be a cornerstone technology for proteomics and biopharmaceutical characterization, enabling insight into protein modifications, interactions, and structures. Recent demands in single-cell proteomics, top-down workflows, and analysis of large native protein assemblies require instruments with higher sensitivity, faster acquisition, and broader mass range coverage.

Objectives and overview of the study

This white paper introduces the Thermo Scientific Orbitrap Ascend Tribrid mass spectrometer and demonstrates how its novel hardware and software advances address key challenges in multiplex quantitative proteomics, native protein analysis up to 800 kDa, and automated instrument calibration. The aims include benchmarking phosphopeptide identification, TMTpro 18plex throughput, extended mass range capability, and user experience enhancements.

Methodology

The study compared the new instrument against previous-generation Tribrid platforms using:

• Phosphopeptide enrichment with Ti-IMAC beads and 90 min LC gradients to evaluate PTM coverage and spectral quality.
• TMTpro 18plex experiments on human cells, analyzing 12 high-pH fractions with 75 min gradients to assess throughput and quantification depth.
• Native MS of purified GroEL (~800 kDa) at 7500 resolution to validate the extended m/z 16 000 mass range.
• Quantitative accuracy tests with the HYPER standard and Real-Time Search–enabled SPS MS3 methods.

Instrumentation

• Ultra-high-field Orbitrap analyzer achieving up to 45 Hz MS/MS at 7500 resolution.
• Next-generation dual-pressure linear ion trap with up to 50 Hz acquisition.
• Dual ion routing multipoles for parallel ion manipulation and faster scan rates.
• QR5 segmented quadrupole for improved precursor isolation and transmission.
• Optional HMRn+ extended mass range up to m/z 16 000.
• Auto-Ready ion source for remote, automated calibration without disrupting LC-MS setup.
• Thermo Scientific EASY-IC/ETD with proton transfer charge reduction and FAIMS Pro Duo interface for gas-phase fractionation.
• Integrated Instrument Control software with customizable, drag-and-drop method templates and Real-Time Search workflows.

Main results and discussion

• Phosphoproteomics: 20 % increase in phosphopeptide identifications and 25 % more confident site localizations compared to Orbitrap Eclipse.
• TMTpro quantification: over 8000 proteins quantified in under 9 hours, a 30 % reduction in analysis time relative to previous platforms.
• Quantitative accuracy: SPS MS3 with Real-Time Search delivered ratios close to expected values (0.6, 1.5, 3.0), outperforming MS2-based methods.
• Native MS: detection of GroEL (~800 kDa) up to m/z 16 000 in a single scan at 7500 resolution, confirming the extended mass range performance.
• Auto-Ready calibration: streamlined remote calibration reduces downtime and ensures consistent performance.

Benefits and practical applications

• High throughput supports large-scale proteomics and single-cell studies with improved sensitivity for low-abundance analytes.
• Enhanced PTM coverage facilitates detailed mapping of phosphorylation and other modifications in signaling studies.
• Extended mass range and multiple fragmentation modes enable comprehensive native and top-down proteoform analysis.
• Automated calibration and intuitive software simplify operation and reduce training requirements for routine QA/QC workflows.

Future trends and potential applications

Emerging applications will leverage this platform for deep multi-omics integration, real-time adaptive acquisition driven by AI, and exploration of novel biotherapeutic modalities. Continued advances in ion mobility, faster algorithms for real-time search, and integration with automated sample preparation will further expand its utility in both research and industrial laboratories.

Conclusion

The Orbitrap Ascend Tribrid mass spectrometer delivers a step change in sensitivity, speed, and versatility, addressing current demands in proteomics, structural biology, and biopharmaceutical analysis. Its combination of innovative hardware and intelligent software equips laboratories to scale up experiments, achieve deeper coverage, and streamline workflows with minimal hands-on time.

References

No formal reference list was provided in the original document.