ExD AQ-25x Option for Agilent LC/Q-TOF - User Guide

Significance of the Topic

The ExD AQ-25x Option enhances Agilent LC/Q-TOF systems by enabling electron-based fragmentation techniques, particularly ECD. This capability complements conventional CID methods, providing richer structural information on peptides, proteins, and labile biomolecules. By preserving post-translational modifications and yielding complementary ion series, ECD facilitates detailed top- and middle-down proteomic analyses and glycan structural elucidation.

Objectives and Overview

This user guide documents the functional design and operation of the ExD AQ-25x Option for Agilent LC/Q-TOF instruments. Key aims include:

• Describing the ExD cell hardware and software architecture.
• Providing step-by-step installation, operation, tuning, and maintenance procedures.
• Introducing fundamental concepts of electron-based fragmentation.

Methodology and Instrumentation

The ExD Option integrates three primary components:

• ExD cell: Compact assembly of permanent magnets and electrostatic lenses replacing the front segment of the collision cell.
• Filament insert and cassette: Rhenium alloy wire filament for thermionic electron emission, housed in a removable cassette.
• ExD Controller and ExDControl software: Supplies lens voltages and filament current according to user-defined profiles.

Operational modes include fly-through transmission (fragment OFF) and ECD (fragment ON). Lens profiles are saved as tunefiles for MS1 and MS2 states. Filament current is optimized (typically 2.4–2.7 A) to balance emission efficiency against filament lifetime.

Key Results and Discussion

Autotuning routines in ExDControl adjust lens voltages to maximize ion transmission or ECD fragment intensities on reference standards (e.g., Agilent tuning mix, amidated substance P). Manual tuning refines electron energy and cloud alignment. Typical ECD efficiencies of 1–5% for doubly charged peptides improve with higher precursor charge states. Supplemental collision energy added post-ExD reduces noncovalent restraints, enhancing sequence coverage. Troubleshooting procedures address filament burnout, current leakage, and connectivity issues.

Benefits and Practical Applications

The ExD AQ-25x Option enables:

• Retention of labile PTMs and improved localization in proteomic workflows.
• Expanded sequence coverage via complementary c-/z-ion series.
• Glycan and glycopeptide structural analysis through cross-ring and glycosidic cleavages.
• Targeted top- and middle-down experiments with high molecular weight species.
• Integration with existing Agilent acquisition and tuning tools for streamlined operation.

Future Trends and Potential Applications

Emerging directions include:

• Integration of EID for singly charged species, expanding small molecule and glycan analysis.
• Development of real-time adaptive tuning to maintain optimal ECD efficiency.
• Advanced bioinformatics pipelines for automated ExD fragment assignment and proteoform mapping.
• Combination with ion mobility separations to deconvolute complex conformational ensembles.

Conclusion

The ExD AQ-25x Option significantly extends the analytical capabilities of Agilent LC/Q-TOF platforms by incorporating robust electron-based fragmentation. Through detailed hardware design, flexible software control, and comprehensive tuning protocols, users can achieve high-resolution structural insights into peptides, proteins, and glycoconjugates. Routine maintenance and troubleshooting guidelines ensure reliability and consistent performance.

References

• U.S. Patents 8,723,113 B2; 9,269,556 B2; 9,305,760 B2; 10,283,335 B2.
• e-MSion ExD AQ-25x Option Site Preparation Checklist.
• e-MSion ExDControl Software User Guide.
• e-MSion ExD Controller User Guide.
• Agilent 6200/6400/6500 LC/MS Maintenance Guide.
• e-MSion ExD AQ-25x Option Quick Start Guide.
• Roepstroff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984.
• Biemann, K. Mass Spectrom. Rev. 1990.
• Domon, B.; Costello, C.E. Glycoconj. J. 1988.