Performance Evaluation of a Modified Quadrupole Orbitrap Mass Spectrometer

Significance of the Topic

The demand for detailed protein characterization in proteomics, biopharma and related research fields drives ongoing enhancements in mass spectrometry instrumentation. High resolution, rapid scan speeds and robust quantification methods are essential to map complex proteomes, support drug development efforts and ensure reliable quality control of biomolecules.

Objectives and Study Overview

This investigation evaluates a modified quadrupole-Orbitrap platform, the Thermo Scientific™ Orbitrap Exploris™ 480, across multiple analytical workflows. The study compares its performance with an established HF-X system, examines data-dependent (DDA) and data-independent (DIA) acquisition modes, explores enhancements from FAIMS ion mobility and TurboTMT processing, and demonstrates intact monoclonal antibody analysis under native and denaturing conditions.

Methodology and Instrumentation

Standardized HeLa and yeast protein digests were analyzed using varying sample loads (50 ng to 1 µg) and chromatographic gradients (5 min to 60 min) on EASY-nLC™ 1200 or Ultimate™ 3000 RSLCnano systems. DDA and DIA acquisitions employed optimized AGC targets, transient lengths and TopN settings. TMT11plex quantification used the embedded TurboTMT option combined with the ΦSDM algorithm for accelerated high-resolution reporter ion processing. Label-free experiments spiked yeast digest into a constant HeLa background to assess quantitation accuracy. Intact monoclonal antibodies were profiled in both native and denatured states to confirm mass and glycoform ratios.

Applied Instrumentation

• Orbitrap Exploris 480 mass spectrometer
• Electrospray source with high-capacity transfer tube and RF-only ion funnel
• Advanced Active Beam Guide and segmented quadrupole for enhanced ion transmission
• Ion routing multipole, curved linear trap and independent charge detector
• Ultra-high vacuum manifold and high-field Orbitrap analyzer (up to 480,000 resolution)
• FAIMS Pro interface
• TurboTMT processing powered by Phase-Constrained Spectrum Deconvolution
• Thermo Scientific Acclaim PepMap RSLC C18 columns

Main Results and Discussion

• In DDA mode using a 60 min gradient, the Exploris 480 matched ~39,000 unique peptides to ~4,800 protein groups per run and ~55,000 peptides to ~5,500 groups overall, equating performance with the Q Exactive HF-X.
• Short gradients (5–13 min) still yielded >6,000 peptides and >1,000 proteins in DDA and identified up to 2,000 proteins in DIA with >80 % quantification and median CV <10 %.
• FAIMS integration provided up to a 24 % increase in identifications at optimal compensation voltage and in-silico combining of multiple CVs enhanced peptide/protein IDs by >35 %.
• TurboTMT application to TMT11plex samples delivered ~30 % more peptide and protein identifications at 15k and 30k MS2 resolutions, leveraging faster ΦSDM processing for high-fidelity reporter quantification.
• Label-free experiments accurately determined yeast spiking ratios (2×, 5×, 10×) with <10 % error and precision improving for higher fold changes.
• Intact mAb analysis under native conditions preserved conformations and charge distributions, while denaturing conditions increased protonation; glycoform ratios were confirmed by accurate mass measurement up to m/z 8,000.

Benefits and Practical Applications

• High-throughput proteome mapping and rapid screening in DDA or DIA workflows
• Enhanced multiplexed quantification with TurboTMT for large-scale comparative studies
• Improved selectivity and depth via FAIMS ion separation
• Reliable label-free quantitation for biomarker discovery and QA/QC assays
• Versatile intact protein profiling for biopharmaceutical characterization

Future Trends and Potential Uses

Emerging directions include deeper integration of ion mobility separators, further acceleration of real-time data processing, coupling high-resolution mass spectrometry with machine learning for automated annotation, expanded native mass workflows for complex assemblies, and miniaturized high-throughput platforms for single-cell proteomics and multi-omics integration.

Conclusion

The Orbitrap Exploris 480 platform combines a compact footprint with robust DDA and DIA performance, advanced ion handling, rapid high-resolution capabilities and enhanced quantification options. It meets current proteomics and biopharma demands for speed, sensitivity and accuracy across diverse analytical scenarios.

References

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