Implementation of Electron-transfer dissociation (ETD) and Electron-transfer/higher-energy collision dissociation (EThcD) on a modified Orbitrap hybrid MS

Significance of the Topic

Electron-transfer dissociation (ETD) and its hybrid variant EThcD (electron-transfer/higher-energy collision dissociation) offer complementary fragmentation pathways to traditional collision-induced methods, generating c- and z-type ions essential for confident peptide sequence assignment, de novo sequencing and labile post-translational modification localization. Their implementation on high-resolution Orbitrap platforms addresses growing demands for fast, sensitive and information-rich proteomic analyses.

Objectives and Study Overview

This work describes the integration of ETD and EThcD on a modified Thermo Scientific Orbitrap Excedion Pro mass spectrometer, systematic optimization of reaction parameters (charge-dependent reaction time scaling and supplemental activation energy) and benchmarking against higher-energy collisional dissociation (HCD) for a complex tryptic HeLa digest.

Methodology and Instrumentation

Sample Preparation and LC-MS Setup:

• Thermo Scientific Pierce HeLa tryptic digest, 20 μg/vial reconstituted in 0.1% formic acid.
• Chromatography: 25 cm × 75 μm Aurora Ultimate column, Vanquish Neo UHPLC, 19.5 min gradient at 200 nL/min.
• Source: EASY-Spray; MS: Orbitrap Excedion Pro in DDA mode, MS1 resolution 60 000, MS2 at 15 000, isolation window Δm/z 1.2, cycle time 1.5 s.

Instrument Modifications for ETD/EThcD:

• Ion Routing Multipole (IRM) upgraded with dual-polarity RF electronics applied to entrance/exit lenses for simultaneous storage of precursor cations and fluoranthene radical anions.
• ETD scan sequence: precursor injection → ETD preparation (polarity switch) → reagent injection → reaction in IRM → optional supplemental collisional activation in C-Trap → Orbitrap detection.

Data Analysis:

• Proteome Discoverer 3.1.1 with custom scripts.
• Performance metrics: Sequest XCorr, high-confidence PSM counts, MS2 scan rate.

Results and Discussion

Optimization of ET(hc)D Conditions:

• Charge-dependent reaction time calibration based on reagent injection time and precursor charge state improved fragmentation efficiency over fixed times.
• Optimal supplemental activation (SA) energy around 30 NCE and reaction time scaling near 100% maximized XCorr scores and PSM confidence.

Benchmarking Against HCD:

• MS2 scan rates: EThcD achieved >15 Hz, comparable to HCD but with richer fragment ion series.
• Cross-correlation (XCorr) scores highest for EThcD (SA = 30, RT scale ≈ 100%), indicating superior spectral quality.
• PSM counts: HCD yielded ~1603 PSMs (XCorr 3.06), EThcD ~1086 PSMs (XCorr 3.68), with EThcD variants balancing depth vs. confidence.

Practical Benefits and Applications

ET(hc)D on Orbitrap Excedion Pro delivers rapid, high-confidence peptide identifications, enhanced sequence coverage for low-charge precursors and reliable localization of labile modifications. The method is well suited for top-down and middle-down proteomics, PTM mapping and challenging de novo sequencing tasks.

Future Trends and Potential Uses

Emerging directions include:

• Real-time adaptive control of reaction times guided by machine learning.
• Integration with advanced ion mobility separations for multidimensional proteomics.
• Expanded applications in glycoproteomics and intact protein analysis.
• Automated calibration routines for seamless method transfer between instruments.

Conclusions

Successful implementation of ETD and EThcD on a modified Orbitrap Excedion Pro enables high-throughput, high-confidence peptide fragmentation. Optimized reaction time scaling and supplemental activation energies yield superior spectral quality compared to HCD, expanding analytical capabilities in proteomics workflows.

Reference

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