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

Significance of the topic

Electron capture dissociation (ECD) on quadrupole time-of-flight (Q-TOF) instruments offers a powerful approach to peptide and protein characterization. The ExD AQ-25x Option extends the capabilities of Agilent 6500 LC/Q-TOF systems by enabling both transmission and fragmentation control in a single cell. This quick start guide outlines essential steps to achieve reliable ECD performance, improving structural analysis of biomolecules and supporting proteomics, QA/QC and industrial analytics applications.

Objectives and Overview of the guide

This guide provides end users with concise instructions for routine setup, autotuning and best practices when operating the ExD AQ-25x Option together with MassHunter Acquisition 11 and ExDControl 3.6. Key goals include:

• Introducing ExDControl software functions and profile management
• Describing autotune and manual tune procedures for MS1 and MS2 transmission
• Detailing ECD tuning protocols for peptide fragmentation
• Recommending best practices to maintain optimal performance

Methodology

The ExD AQ-25x Option is managed by two software components: MassHunter Acquisition for instrument control and ExDControl for the ExD cell lenses and filament currents. Users select and load voltage profiles for MS1 (total ion) and MS2 (isolation) modes. Autotune methods—coarse, refine and extended—automatically optimize lens voltages based on predefined mass lists (e.g., Tunemix, Substance P). Manual tuning adjustments focus on fine-tuning filament current (typical starting point 2.4 A) and verifying compatibility with main ion optics.

Instrumentation used

• Agilent 6500 LC/Q-TOF system equipped with ExD AQ-25x Option
• MassHunter Acquisition software version 11
• ExDControl software version 3.6

Main procedures and discussion

1. MS1 Transmission Tuning
   • Load MS1 profile and set initial parameters; infuse Tunemix in Total Ion mode.
   • Run ExDControl refine tune, adjust filament current in 0.05 A steps to maximize signal.
   • Perform MassHunter system tune with current parameters, high-resolution TOF slicer.
   • Optionally refine MS1 profile again on Tunemix.
2. MS2 Transmission Tuning
   • After MS1 setup, isolate m/z 922 in MassHunter and perform ExD refine tune for MS2.
3. MS2 ECD Tuning
   • Infuse Substance P and isolate parent m/z 674.
   • Select ECD mass list and fragments sub-list; run ExD refine tune.
   • Adjust filament current as needed to enhance ECD fragment abundance.

Benefits and practical applications

• Enhanced molecular coverage: ECD preserves labile modifications and provides complementary fragment ions.
• Streamlined workflows: automated autotune methods reduce setup time and user intervention.
• Improved reproducibility: standardized profiles and best practices ensure consistent performance day-to-day.
• Versatility: supports routine QA/QC, proteomics research, top-down and middle-down experiments.

Future trends and applications

Looking ahead, integration of ExD with real-time data analysis and AI-driven tuning promises faster optimization and adaptive workflows. Expanded use in large-scale proteomics, biologics characterization and PTM mapping will benefit from improved cell designs and extended mass range. Automation and remote diagnostics will further streamline maintenance and ensure continuous high-throughput operation.

Conclusion

This quick start guide equips users with a clear roadmap for deploying the ExD AQ-25x Option on Agilent 6500 LC/Q-TOF platforms. By following prescribed tuning procedures and best practices, laboratories can harness reliable transmission and high-quality ECD fragmentation to advance biomolecular analysis.

Reference

• ExD AQ-25x Option User Guide
• ExDControl Software User Guide
• Application Note: Top-down Disulfide Mapping, Revision R001, July 2021
• Application Note: Modification Localization, Revision R002, May 2022
• J. S. Beckman et al., Improved Protein and PTM Characterization with a Practical Electron-Based Fragmentation on Q-TOF Instruments, J. Am. Soc. Mass Spectrom., 2021, doi:10.1021/jasms.0c00482.