Increasing the depth of single shot proteomics with enhanced data acquisition and processing strategies

Significance

Single-shot proteomics aims to identify and quantify thousands of proteins in a single liquid chromatography–mass spectrometry (LC-MS) run. Improved depth of proteome coverage enhances our ability to study complex biological systems, reduces analysis time, and supports high-throughput applications in research, clinical laboratories, and pharmaceutical quality control.

Study Objectives and Overview

This study evaluated how optimized data acquisition parameters combined with an advanced search algorithm can deepen single-shot proteome coverage. Key goals included:

• Determining the optimal MS2 isolation window for both Orbitrap (OT/OT) and ion-trap (OT/IT) fragmentation modes.
• Assessing the impact of maximum injection time (MaxIT) on peptide and protein identifications.
• Exploring the benefit of gas-phase fractionation using the FAIMS Pro Duo interface across multiple compensation voltages (CVs).
• Comparing standard processing (Sequest HT with INFERYS rescoring) to the CHIMERYS intelligent search algorithm within Proteome Discoverer 3.0.

Methodology and Instrumentation

Sample Preparation and Chromatography:

• Thermo Scientific™ Pierce™ HeLa Digest Standard reconstituted in 5% acetonitrile/0.1% formic acid.
• Separation on a µPac Neo column at 300 nL/min using a Vanquish Neo UHPLC system with gradients from 30 minutes to 3 hours.

Mass Spectrometry:

• Thermo Scientific™ Orbitrap Eclipse™ Tribrid mass spectrometer operated in data-dependent acquisition mode.
• Full scans: OT at 60,000 resolution; OT/IT with 240,000 full scans and Turbo-IT MS2.
• Quadrupole isolation widths varied between 0.4 Th and 3 Th for MS2.
• MaxIT settings tested: 10–25 ms for OT/IT; 11–35 ms for OT/OT.
• FAIMS Pro Duo interface evaluated with two CVs (–50, –70) and three CVs (–40, –60, –80).

Data Processing:

• Proteome Discoverer 3.0 workflows: Sequest HT + INFERYS rescoring versus CHIMERYS + Percolator.
• Performance metrics: unique peptide and protein group counts, PSMs per spectrum, total MS2 coverage.

Main Results and Discussion

Optimization of Isolation Window and MaxIT:

• Wider MS2 windows (2–4 Th for OT/OT; 1.2–1.5 Th for OT/IT) increased identifications by up to 15% relative to narrower windows.
• Increasing MaxIT to 22 ms (OT/OT) and 15 ms (OT/IT) yielded marginal gains; longer injection times (>25 ms) reduced throughput and identifications due to fewer spectra acquired.

Impact of CHIMERYS Search Algorithm:

• CHIMERYS processing provided 20–35% more unique peptides and protein groups compared to Sequest HT + INFERYS across all gradient lengths and sample loads.
• PSMs per MS2 spectrum increased by ~20% with CHIMERYS, reflecting improved deconvolution of chimeric spectra.

FAIMS-Enhanced Gas-Phase Fractionation:

• Single-shot runs with individual FAIMS CVs identified ~84,000 unique peptides; combining three CVs raised identifications to ~110,000 (+31%).
• FAIMS fractionation complemented wide-window acquisition and CHIMERYS processing, delivering the deepest coverage in single runs.

Benefits and Practical Applications

• Higher proteome coverage in single injections reduces total instrument time and sample consumption.
• Wide-window acquisition combined with CHIMERYS enables rapid, high-depth analyses suitable for biomarker discovery, clinical proteomics, and QA/QC workflows.
• FAIMS gas-phase fractionation adds orthogonal selectivity, unlocking low-abundance and co-eluting peptides without extended gradients.

Future Trends and Opportunities

• Integration with data-independent acquisition (DIA) and machine-learning–driven real-time acquisition adjustments.
• Expanded use of intelligent algorithms for automated parameter tuning and on-the-fly spectrum deconvolution.
• Development of novel ion mobility technologies and CV scheduling strategies for even deeper single-shot coverage.

Conclusion

Combining optimized MS2 isolation widths, finely tuned injection times, FAIMS-based gas-phase fractionation, and the CHIMERYS intelligent search algorithm substantially enhances single-shot proteome depth. This integrated approach delivers faster, more comprehensive analyses, supporting large-scale proteomics studies and routine laboratory applications.

References

No formal references were provided in the source document.