Improving high-throughput Top-Down proteomics using a modified hybrid quadrupole-ultra-high-field-Orbitrap mass spectrometer

Importance of the topic

Top-Down proteomics enables the direct analysis of intact proteins and their variants, providing comprehensive information on proteoforms that bottom-up approaches cannot deliver. High-throughput Top-Down workflows are critical for large-scale studies in structural biology, biopharmaceutical characterization, and biomarker research. By improving instrument performance and data acquisition strategies, researchers can identify low-abundance proteoforms more confidently and increase overall proteome coverage.

Objectives and study overview

This study aimed to demonstrate enhanced high-throughput Top-Down proteomics using a modified hybrid quadrupole-ultra-high-field-Orbitrap mass spectrometer (Q Exactive HF-X) combined with advanced data-dependent acquisition (DDA) algorithms. Key goals included reducing redundant fragmentation of abundant species, improving signal-to-noise ratios for intact proteins, and increasing the number and diversity of proteoforms identified in complex samples.

Methodology and instrumentation

A series of hardware and software upgrades were implemented on the Q Exactive HF platform to create the HF-X instrument. These included:

• A brighter ion source interface featuring a High-Capacity Transfer Tube and an electrodynamic ion funnel for enhanced ion transmission.
• On-the-fly charge state assignment and deconvolution algorithms to extract useful frequency information from time-domain beat patterns of isotopic envelopes.
• Optimized Top-Down DDA workflows named Medium-High and High-High, employing sliding isolation windows, single-charge-state selection, and optimum collision energies for HCD fragmentation.

Reversed-phase chromatographic separation of intact proteins was performed on a MabPac-RP column using a Vanquish Horizon UHPLC system. Data processing and identification were achieved through Thermo Scientific BioPharma Finder, Proteome Discoverer, and ProSight PD software.

Main results and discussion

Comparison of transient lengths demonstrated that a 16 ms setting at 7,500 resolution provided a threefold increase in signal-to-noise for intact Carbonic Anhydrase II versus 32 ms transients. The improved DDA workflow reduced redundant MS/MS events for abundant proteoforms, enabling deeper sampling of lower-abundance species. In E. coli extract analyses, the modified HF-X instrument and High-High method identified 35% more proteoforms than the standard HF platform. For purified E. coli 40S and 60S ribosomal protein samples, the combined Medium-High and High-High approaches achieved unambiguous identification of all expected ribosomal proteins (4.4–61 kDa) in a single 90-minute LC–MS/MS run.

Benefits and practical applications

The enhanced HF-X system offers:

• Higher throughput through rapid full MS scans at 7,500 resolution without compromising sensitivity.
• Intelligent acquisition that minimizes wasted MS/MS scans on redundant precursors.
• Extended dynamic range allowing detection of low-level proteoforms alongside highly abundant species.

These improvements support applications in proteoform-level biomarker discovery, biopharmaceutical quality control, and routine laboratory workflows requiring high accuracy mass measurements and confident identification of intact proteins.

Future trends and opportunities

Further development of real-time data analysis algorithms and adaptive acquisition schemes is expected to push Top-Down throughput even higher. Integration with ion mobility separation, AI-driven precursor prioritization, and multiplexed fragmentation strategies may unlock deeper proteome coverage. As instrument sensitivity advances, clinical and industrial laboratories will adopt Top-Down proteomics for routine characterization of complex protein assemblies and post-translational modifications.

Conclusion

This work highlights significant gains in Top-Down proteomics from combined hardware enhancements and optimized DDA workflows on a hybrid quadrupole-ultra-high-field-Orbitrap platform. By strategically reducing redundant fragmentation and extracting maximal information from intact protein signals, the improved HF-X system achieves faster, more sensitive, and more comprehensive proteoform identification, paving the way for broader adoption of high-throughput Top-Down methodologies in research and industrial settings.