Optimization of RP-LC-MS Top-down Protein Analysis on an Orbitrap Fusion Lumos Tribrid MS with the Advanced Peak Determination Algorithm

Importance of the Topic

The direct analysis of intact proteoforms by top-down LC-MS plays a critical role in characterizing protein isoforms, post-translational modifications and sequence variants without prior digestion. However, low-resolution MS1 detection dilutes signal across multiple charge states and complicates real-time charge assignment, limiting data-dependent acquisition efficiency and proteome coverage.

Goals and Overview of Study

This work evaluates the impact of an Advanced Precursor Determination (APD) algorithm on Orbitrap Fusion Lumos Tribrid mass spectrometry for top-down proteomics. The objectives are to minimize redundant MS2 sampling of the same protein, improve precursor charge state assignment in low-resolution scans, and extend depth of analysis in both simple (intact protein standards) and complex (E. coli lysate) samples.

Methods and Instrumentation

Sample preparation and instrument setup were as follows:

• Pierce Intact Protein Standard Mix (six proteins, 9–68 kDa) and E. coli lysate reconstituted in 0.1% formic acid.
• Vanquish UHPLC with MAbPac RP column (10 cm) at 200 µL/min, 20 min gradient for standards and 60 min for lysate.
• Orbitrap Fusion Lumos Tribrid MS with APD enabled, operating in “low-high” (MS1 at 15 000 FWHM, MS2 at 120 000 FWHM) and “high-high” (MS1 and MS2 at 120 000 FWHM) modes.
• Data processing using Proteome Discoverer 2.2 with ProSightPD templates and E. coli database.

Main Results and Discussion

Key findings include:

• APD accurately assigns charge states across entire envelopes in real time, enabling MS2 triggering on true intact proteoform signals rather than chemical noise.
• Balanced MS1/MS2 scan distribution demonstrated for protein standards; legacy algorithms required “allow unassigned charge states” and yielded unbalanced acquisition.
• In Pierce standard mixture, APD delivered near tenfold increase in unique proteoform identifications in low-high experiments and over 10% gains in high-high mode compared to standard SPD filtering.
• In E. coli lysate, APD preserved MS2 triggering under stringent charge-state filters in both acquisition schemes, enhancing sampling depth in complex proteomes.

Benefits and Practical Application

The APD algorithm streamlines top-down data-dependent workflows by:

• Reducing redundant sampling of multiple charge states of the same proteoform.
• Improving charge state assignment directly in low-resolution MS1 scans.
• Increasing the number of informative MS/MS events and unique protein identifications.
• Enhancing throughput and sensitivity in both simple standards and complex biological samples.

Future Trends and Possibilities of Use

Emerging directions include:

• Integration of APD with advanced fragmentation methods (ETD, HCD, UVPD) for richer structural information.
• Application to higher-molecular-weight proteomes and native top-down workflows.
• Development of more sophisticated data-dependent filters and real-time decision engines.
• Coupling with machine-learning algorithms for improved proteoform annotation and quantitation.

Conclusion

The Advanced Precursor Determination algorithm on the Orbitrap Fusion Lumos significantly enhances top-down LC-MS performance by enabling accurate, real-time charge state assignment in low-resolution full scans. This innovation reduces redundant MS2 events, deepens proteoform coverage, and offers a robust platform for detailed intact protein analysis.