Optimizing Top Down Analysis of Proteins on Orbitrap Fusion Lumos Tribrid Mass Spectrometer

Significance of the Topic

Top-down mass spectrometry of intact proteins delivers comprehensive sequence information, including post-translational modifications and proteoforms, critical for biopharmaceutical quality control, biomarker discovery and fundamental protein research.

Objectives and Study Overview

This study aimed to optimize top-down proteomic analysis on the Orbitrap Fusion Lumos Tribrid™ mass spectrometer. Key goals were to maximize sequence coverage, improve acquisition speed, and leverage advanced vacuum and ETD HD fragmentation to enhance detection of large intact proteins and glycoproteins.

Methodology and Instrumentation

Sample Preparation

• Protein standards (Ubiquitin, Carbonic anhydrase, Enolase) at 1–4 pmol on column; intact IgG and recombinant Trop2 protein reduced with TCEP, deglycosylated with PNGase F.
• Proteins loaded onto a ProSwift monolithic capillary column (200 µm × 25 cm) via a Thermo Scientific UltiMate 3000 RSLCnano system operated in microflow mode.

Mass Spectrometry

• Orbitrap Fusion Lumos Tribrid equipped with Advanced Vacuum Technology to allow elevated multipole pressure (1–2 mtorr) while maintaining ultra-high vacuum in the Orbitrap.
• Fragmentation modes: ETD HD (high dynamic range), standard ETD, HCD and CID. AGC targets: 1e6 for ETD HD, 3e5 for ETD reagent.
• Acquisition resolutions: 240 K for direct infusion, 120 K for LC-MS experiments; reaction times 10–20 ms; collision energy optimized for HCD/CID.
• Data processed with ProSightPC™ 3.0 SP1 software with 15 ppm fragment tolerance.

Key Results and Discussion

Direct Infusion

• Ubiquitin: 76 % sequence coverage with ETD HD vs. 65 % with standard ETD.
• Carbonic anhydrase: rapid acquisition on Fusion Lumos yielded higher coverage in shorter time compared with the original Fusion platform.
• IgG light chain: 56 % coverage (ETD HD) vs. 48 % (ETD); combining all fragmentation modes delivered 91 % light-chain and 63 % heavy-chain coverage.

Online LC-MS

• Carbonic anhydrase: 68 % coverage; Enolase: 41 % coverage by merging ETD HD, HCD and CID spectra.
• Trop2 glycoprotein: base-peak LC chromatogram resolution at 15 K revealed clear elution and isotopic profiles; top-down fragmentation confirmed an N-terminal truncation to residue 30 (vs. predicted 26), pyroglutamate formation at the new N-terminus, and deamidation at three asparagine sites after PNGase F treatment.
• Intact IgG by LC-MS ETD HD achieved 20 % light and 18 % heavy chain coverage in 5 µscan acquisitions.

Benefits and Practical Applications

• Enhanced sequence coverage at shorter acquisition times enables high-throughput proteoform characterization.
• Advanced ETD HD mode improves dynamic range for large proteins, facilitating detection of low-abundance fragments.
• Multi-mode fragmentation strategy supports near-complete mapping of antibodies and complex glycoproteins, valuable for biotherapeutic QA/QC.

Future Trends and Potential Applications

• Integration with faster chromatography and automation to scale top-down workflows for proteome-wide studies.
• Further development of data analysis software to automate proteoform identification and PTM localization.
• Application in clinical proteomics for detailed profiling of disease-relevant protein variants and therapeutic monitoring.

Conclusion

Optimized use of ETD HD on the Orbitrap Fusion Lumos Tribrid significantly enhances sequence coverage and throughput for intact protein analysis. Combining ETD HD, ETD, HCD and CID yields comprehensive proteoform maps across a range of model and glycosylated proteins, demonstrating strong potential for advanced biopharmaceutical and proteomic applications.

Instrumentation Used

• Thermo Scientific Orbitrap Fusion Lumos Tribrid Mass Spectrometer
• Thermo Scientific UltiMate 3000 RSLCnano System
• ProSwift Monolithic Capillary Column (200 µm × 25 cm)
• ProSightPC™ 3.0 SP1 Software

References

1. Thermo Scientific™ Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer Product Specifications.
2. C. Mullen et al., Considerations for Improved ETD Performance in Top-Down Applications, Poster 48 (2015).
3. J. Canterbury et al., Improvements for High-Resolution Analysis on a Modified Tribrid Mass Spectrometer, Poster 124 (2015).