Ion Pre-Accumulation for High Speed Orbitrap Exploris Operation

Importance of the Topic

Orbitrap mass spectrometers are essential tools in proteomics and high-resolution analysis. However, their maximum scan rates have been constrained by preparation overheads in the C-Trap and Ion Routing Multipole (IRM), limiting overall duty cycle to below 50% at high repetition rates. Enhancing ion utilization during these dead times can dramatically improve sensitivity, throughput, and depth of coverage in complex biological analyses.

Objectives and Study Overview

This study aimed to implement and evaluate a pre-accumulation scheme in a modified Orbitrap Exploris 480 to eliminate dead time during C-Trap/IRM operations. Key goals included:

• Developing firmware modifications to trap ions in the bent flatapole during analyzer overhead.
• Comparing sensitivity and scan performance with and without pre-accumulation.
• Assessing the impact on protein and peptide identifications in data-dependent acquisition (DDA) of HeLa digest.

Used Instrumentation

The following instrumentation was employed:

• Thermo Scientific™ Orbitrap Exploris™ 480 mass spectrometer (modified firmware).
• Bent flatapole quadrupole ion guide with switchable exit lens potentials.
• Thermo Scientific™ Vanquish™ Neo UHPLC and EASY-nLC™ systems for nano-LC separations.
• Thermo Scientific™ Pierce™ FlexMix™ Calibration Solution and MRFA peptide infusion via syringe pump.
• Thermo Scientific™ Proteome Discoverer™ 3.0 with Sequest HT and Percolator for data analysis.

Methodology and Instrumentation

The pre-accumulation concept relies on alternating the bent flatapole exit lens between trapping (+10 V) during the C-Trap/IRM overhead (~10 ms) and transmitting (–35 V) for ion injection. Firmware was patched to control these voltage switches independent of automatic gain control, enforcing a fixed injection time. Experiments included:

• Infusion of MRFA peptide to quantify signal changes across 2–16 ms injection times and 47–76 Hz repetition rates.
• DDA runs of 200 ng HeLa digest across gradients of 7.6, 14.4, 24.6, and 60 min at 40 Hz (standard) and 70 Hz (pre-accumulation).
• Assessment of signal-to-noise, peptide spectrum matches (PSMs), unique peptide and protein identifications.

Main Results and Discussion

Pre-accumulation yielded more than a twofold increase in MS/MS signal intensity while preserving fragment relative ratios. At an 8 ms Orbitrap transient (3 ms inject), duty cycle (>70 Hz) was maintained with minimal loss in ion current. HeLa DDA analysis showed:

• ~7% increase in matched MS2 spectra with pre-accumulation on short gradients.
• 8% more unique peptides and 5% more proteins identified under 70 Hz operation compared to 40 Hz standard methods.
• Greatest gains observed in high-throughput, short-gradient experiments.

Benefits and Practical Applications

The adaptation significantly enhances throughput and sensitivity in proteomics workflows. Laboratories seeking rapid, deep proteome coverage—such as clinical biomarker discovery and industrial QA/QC—can leverage higher scan frequencies without sacrificing data quality. The approach is compatible with existing UHPLC setups and common data analysis pipelines.

Future Trends and Potential Applications

Further development may focus on integrating pre-accumulation with dynamic AGC control, extending the method to data-independent acquisition (DIA), and applying it across other Orbitrap platforms. Real-time adjustment of trapping timing based on chromatographic profiles could further optimize ion utilization, enabling ultrafast clinical proteomics and large-scale biomarker screening.

Conclusion

Pre-accumulation in the bent flatapole of a modified Orbitrap Exploris 480 overcomes the duty-cycle limitations imposed by C-Trap/IRM overhead. This simple firmware-based scheme doubles MS/MS signal and enables stable operation at up to 70 Hz, translating into measurable gains in peptide and protein identifications, especially under high-throughput conditions.

Reference

• Arrey TN, Stewart H, Harder A. Ion Pre-Accumulation for High Speed Orbitrap Exploris Operation. Thermo Fisher Scientific; 2022.