A Novel Instrumental Strategy for Parallel Reaction Monitoring of Intact Proteins with an Orbitrap Analyzer

Importance of the Topic

Top-down proteomics enables detailed characterization of intact proteins, including post-translational modifications and isoforms. However, conventional quadrupole isolation of a single narrow m/z window leads to significant sensitivity loss when targeting high-mass proteins spread across multiple charge states. A more efficient precursor selection strategy is essential to boost signal intensity, throughput, and confidence in protein identification.

Objectives and Study Overview

This study aims to introduce and validate a novel parallel reaction monitoring approach for intact proteins using an Orbitrap analyzer. The core objective is to demonstrate that a time‐of‐flight (TOF) mass selector can concurrently isolate multiple m/z windows corresponding to different charge states of a target protein, followed by higher‐energy collisional dissociation (HCD) and high-resolution Orbitrap detection. Both simulation and initial experimental results are presented to prove feasibility.

Methodology

• Three‐stage TOF filtering concept:

• Deflection gate for coarse pre‐selection of a broad m/z range.
• “Bouncer” pulsed plates for fine selection of multiple discrete m/z windows within the pre‐selected range.
• Deceleration and transfer of selected ions into the C‐trap and HCD cell for accumulation and fragmentation.

• Sample preparation involved calibration mixtures (n‐butylamine, caffeine, MRFA peptide, Ultramark) in 50/50 water/acetonitrile with 0.1% acetic acid and infusion of ubiquitin solutions by syringe pump.

Used Instrumentation

• Modified Thermo Scientific Q Exactive Orbitrap with H‐ESI II source.
• Orthogonal linear TOF separator capable of selecting ions up to m/z 20 000 at resolution ~100.
• Injection flatapole mass filter and quadrupole collision cell introduced upstream of the TOF region.
• Hardware modifications: reduced RF multipole frequencies, increased HCD cell gas flow, custom printed circuit boards.
• Software updates to the Tune program for control of pulsed TOF gating and timing.

Main Results and Discussion

Simulations of ion trajectories and energy distributions confirmed that the three‐stage gating achieves high‐resolution selection of multiple m/z windows with minimal transmission losses. Experimental panoramic mass spectra of the calibration mixture showed clear isolation of two parallel windows compared to a full‐scan acquisition. Infusion of 1 µM ubiquitin yielded enhanced signal‐to‐noise for selected charge states, validating the concept. Energy distribution measurements demonstrated efficient ion focusing and accumulation in the C‐trap and HCD cell, supporting robust downstream fragmentation.

Benefits and Practical Applications

• Up to an order‐of‐magnitude increase in sensitivity for top‐down and targeted protein analyses.
• Simultaneous interrogation of multiple charge states in a single scan enhances throughput and reduces cycle time.
• Broad applicability to targeted workflows, including PRM, MRM, and data‐independent acquisition for intact proteins.
• Lays groundwork for extending MS3 strategies to intact protein complexes and subunit analysis.

Future Trends and Possibilities

Continued optimization will focus on maximizing ion transmission during TOF extraction, expanding to more than two parallel selection windows, and adapting the concept to native MS of large protein assemblies. Integration with high‐pressure trapping and fine‐tuning of pulsing parameters may further enhance duty cycle and mass coverage. Ultimately, full MS3 implementation for complex top‐down experiments will enable comprehensive characterization of protein assemblies and biomolecular interactions.

Conclusion

This study demonstrates the feasibility of a novel TOF‐based parallel precursor selection strategy on an Orbitrap platform. Both simulations and initial experiments confirm significant gains in sensitivity and identification confidence for intact protein analysis. The approach promises transformative improvements in top‐down proteomics and targeted mass spectrometry workflows.

Reference

1. A. Catherman et al., Biochem. Biophys. Res. Commun., 445(4), 2014, 683–693.
2. M. Belov et al., Anal. Chem., 85(23), 2013, 11163–11173.