Identification of Amino Acid Isomers Using Electron Capture Dissociation in the Agilent 6545XT AdvanceBio LC/Q-TOF System

Importance of the Topic

Distinguishing between isobaric amino acids such as leucine and isoleucine or aspartate and isoaspartate is critical for accurate protein characterization, with implications for understanding protein structure, function, aging, and disease mechanisms. Conventional collision-based fragmentation techniques cannot resolve these isomers, motivating the adoption of electron-based dissociation methods.

Objectives and Overview of the Study

This application note evaluates the Agilent 6545XT AdvanceBio LC/Q-TOF system equipped with an ExD electron capture dissociation cell and ExDViewer software for unambiguous identification of amino acid isomers. Performance was demonstrated on a synthetic peptide (REALLYisoDELIGHTFLK) and intact bovine ubiquitin to showcase peptide and top-down protein analysis workflows.

Použitá metodika a instrumentace

Samples were directly infused using a New Era model 300 syringe pump at 20 µL/min via PEEK tubing into the Dual Jet Stream electrospray source. Electron capture dissociation parameters were optimized in ExDControl using melittin/tune mix for MS1 transmission and melittin or ubiquitin fragment lists for MS2. Targeted acquisition methods were configured in MassHunter Acquisition for precursor isolation. Fragment spectra were processed with ExDViewer’s targeted deconvolution presets, enabling iterative multi-pass matching and intuitive annotation of side-chain fragments and isoAsp ions.

Použitá instrumentace

• Agilent 6545XT AdvanceBio LC/Q-TOF mass spectrometer
• Agilent ExD electron capture dissociation cell
• Agilent Dual Jet Stream electrospray ionization source
• New Era model 300 syringe pump
• Agilent ExDControl software v3.6
• Agilent MassHunter Acquisition software v11.0
• Agilent ExDViewer software v4.5.14

Main Results and Discussion

Electron capture dissociation generated diagnostic w-type fragments that differentiate leucine (loss of 43 Da) from isoleucine (loss of 29 Da), and c + 57/ z – 57 ions for isoaspartate detection. In the synthetic peptide workflow, pure ECD spectra revealed all five Leu/Ile sites and the engineered isoAsp, whereas CID spectra lacked these distinguishing features. Pre-activation collisional energies reduced fragment intensities, underscoring the value of ECD only. In top-down analysis of ubiquitin (11+ precursor), ECD achieved 99 % sequence coverage, resolved 10 of 16 Leu/Ile isomers, and detected isoAsp at positions 39 and 52 through ExDViewer’s interactive sequence mapping.

Benefits and Practical Applications

This approach offers rapid, high-confidence differentiation of isobaric residues in peptides and intact proteins, reducing preparatory steps and artifact risk. It enhances sequence assignment accuracy, supports comprehensive proteoform characterization, and addresses critical needs in biopharmaceutical research, quality control, and structural proteomics.

Future Trends and Potential Applications

Wider adoption of electron-based fragmentation on Q-TOF platforms is anticipated, alongside automated tuning, integration with machine learning for spectral interpretation, expanded mapping of post-translational modifications, and deployment in complex proteomics workflows and therapeutic antibody development.

Conclusion

The combination of ECD on the Agilent 6545XT AdvanceBio LC/Q-TOF and intuitive ExDViewer analysis establishes a robust platform for resolving amino acid isomers, offering comprehensive coverage and clear identification where traditional CID methods fall short.

Reference

1. Kjeldsen F Haselmann KF Sørensen ES Zubarev RA Distinguishing of Ile/Leu Residues in PP3 Protein by Hot Electron Capture Dissociation in FTICR MS Anal Chem 2003 75 6 1267–1274
2. Wakankar AA Borchardt RT Eigenbrot C Shia S Wang YJ Shire SJ Liu JL Aspartate Isomerization in Complementarity Determining Regions of Two Monoclonal Antibodies Biochemistry 2007 46 6 1534–1544
3. Wang J Mukherjee S Zubarev RA Isoaspartate and Neurodegeneration Aging 2022 14 22 8882–8883
4. Geiger T Clarke S Deamidation Isomerization Racemization at Asparaginyl and Aspartyl Residues in Peptides J Biol Chem 1987 262 2 785–794
5. Yang H Zubarev RA Mass Spectrometric Analysis of Asparagine Deamidation and Aspartate Isomerization in Polypeptides Electrophoresis 2010 31 1764–1772
6. Han H Xia Y McLuckey SA Ion Trap Collisional Activation of c/z Ions from Gas-Phase Ion/Ion ETD J Proteome Res 2007 6 8 3062–3069
7. Beckman JS Voinov VG Hare M Sturgeon D Vasil’ev Y Oppenheimer D Shaw JB Wu S Glaskin R Klein C et al Improved Protein and PTM Characterization with Practical Electron-Based Fragmentation on Q-TOF Instruments J Am Soc Mass Spectrom 2021 32 8 2081–2091