Advancing phosphorylation, ADMA, and O-GlcNAc PTM analysis with HCD and EThcD on the Orbitrap Excedion Pro mass spectrometer

Significance of the topic

The analysis of post-translational modifications (PTMs) such as phosphorylation, asymmetric dimethylarginine (ADMA), and O-linked glycosylation (O-GlcNAc) is essential for understanding cellular signaling, disease mechanisms, and protein function regulation. These PTMs are often low-abundance, labile, and distributed across many possible sites, creating analytical challenges that require high sensitivity, robust fragmentation strategies, and specialized software to achieve confident peptide identification and precise site localization.

Goals and study overview

This technical note evaluates the performance of the Thermo Scientific Orbitrap Excedion Pro hybrid mass spectrometer for in-depth PTM analysis. The study demonstrates how combining higher-energy collisional dissociation (HCD) and electron-transfer/higher-energy collisional dissociation (EThcD) improves detection, sequence coverage, and site localization across phosphorylation, ADMA, and O-linked glycopeptides. The work also illustrates an acquisition strategy (HCD product-dependent EThcD) that selectively triggers EThcD for glycopeptide candidates to increase throughput and dynamic range.

Methodology

Samples: Mouse liver and brain lysates and human cancer cell lines (HCT116, MKN-45) were digested with trypsin or LysC. Enrichment employed Cell Signaling Technology PTMScan kits for phosphotyrosine, ADMA, and O-GlcNAc, and global phosphopeptide enrichment used IMAC Fe-NTA beads.

Acquisition: Data-dependent acquisition (DDA) was used with automated per-precursor optimization of fragmentation mode. HCD and EThcD acquisition modes were compared; for O-glycopeptides an HCD-pd-EThcD method was applied where HCD MS2 spectra were screened for oxonium ions and only flagged precursors triggered EThcD scans.

Data processing: Phosphopeptide and ADMA datasets were processed with Proteome Discoverer or FragPipe/MSFragger; O-GlcNAc data were analyzed with Byonic and O-Pair (O-Pair leverages HCD for composition and EThcD for intact-glycan backbone fragments to assign sites). Results were filtered to 1% precursor and protein-group FDR and visualized in R.

Instrumentation used

• Thermo Scientific Orbitrap Excedion Pro hybrid mass spectrometer (ETD-capable)
• Thermo Scientific Vanquish Neo UHPLC system with Ion Opticks Aurora Ultimate 25×75 XT C18 column and EASY-Spray source
• PTMScan enrichment kits and IMAC Fe-NTA magnetic beads (Cell Signaling Technology)
• Consumables: LC-MS grade water, acetonitrile and formic acid
• Software: Proteome Discoverer 3.2, MSFragger/FragPipe, Byonic 5.2.5, O-Pair (MetaMorpheous), R/RStudio for visualization

Main results and discussion

Phosphorylation

• IMAC-enriched samples produced deep coverage: 5,892 unique phosphosites identified across HCD and EThcD analyses.
• Phosphotyrosine enrichment (PTMScan HS kit) yielded 2,941 unique phosphotyrosine sites. Fragmentation-mode complementarity was observed: ~40% of pTyr sites were unique to HCD, ~15% unique to EThcD, with remaining sites identified by both methods.
• EThcD improved site localization in cases where HCD spectra lacked sufficient fragment-ion coverage near the modification site, demonstrating the practical value of electron-driven fragmentation for ambiguous assignments.

ADMA (asymmetric dimethylarginine)

• ADMA-enriched samples produced nearly 300 confidently localized ADMA sites.
• Although HCD returned a larger total peptide count, EThcD provided more ADMA-specific peptide-spectrum matches (PSMs): EThcD yielded 798 ADMA PSMs versus 555 by HCD (total PSMs: EThcD 5,714; HCD 7,850), indicating better targeted sensitivity for this modification with EThcD.

O-linked glycosylation (O-GlcNAc)

• O-glycopeptides are labile under collisional activation; EThcD preserves intact glycan-bearing backbone fragments enabling confident site localization even on multiply glycosylated peptides.
• The HCD-pd-EThcD acquisition (oxonium-ion-triggered EThcD) improved duty cycle and dynamic range by avoiding unnecessary EThcD scans while ensuring glycopeptide precursors received informative ETD-type fragmentation.
• Integration with O-Pair enabled a combined evidence scoring approach (HCD for composition, EThcD for intact glycan-containing fragments) resulting in accurate assignment of multiple glycosylation sites in challenging sequences (examples include peptides with up to 12 potential O-glycosylation sites where EThcD enabled correct localization that stepped-HCD misassigned).

Benefits and practical applications

• Complementary fragmentation: Combining HCD and EThcD expands identifiable PTM space and improves confidence in site localization, important for signaling studies and biomarker discovery.
• Targeted productivity: HCD-pd-EThcD reduces analysis overhead for glycoproteomics by selectively triggering costly EThcD scans only when glycan diagnostic ions are detected, increasing throughput for complex samples.
• Sensitivity and breadth: The Excedion Pro system enabled identification of thousands of modification sites across enrichment strategies, demonstrating suitability for large-scale PTM profiling in research and applied settings (e.g., signaling pathway mapping, disease-related PTM studies, targeted PTM discovery).

Future trends and potential applications

Advances likely to accelerate PTM research include tighter integration of intelligent acquisition strategies (real-time spectral triage and product-dependent triggering), further optimization of ETD/EThcD reaction parameters for low-charge or low-abundance precursors, and enhanced computational frameworks that integrate multiple fragmentation types for probabilistic site localization. Application areas that will benefit include phospho-signaling pathway mapping, cardiovascular and renal disease biomarker discovery linked to ADMA, and comprehensive O-glycoproteome mapping in immunology and cancer research. Automation of enrichment workflows and improved hybrid search engines that unify HCD and ETD evidence will also increase throughput and reliability.

Conclusion

The Orbitrap Excedion Pro mass spectrometer, when operated with a strategy combining HCD and EThcD (including HCD-pd-EThcD for glycopeptides), delivers enhanced sensitivity, complementary fragmentation coverage, and more confident PTM site localization across phosphorylation, ADMA, and O-glycosylation chemistries. EThcD in particular increases sequence coverage and accurate localization for labile or ambiguous modification sites, while product-dependent triggering improves throughput for glycoproteomics. Together these capabilities support deeper proteome and PTM characterization in complex biological samples.

References

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