Phosphorylation, ADMA, and O-GlcNAc Analysis on the Orbitrap Excedion Pro Mass Spectrometer

Post-translational modifications (PTMs) are where so much of biology’s complexity lives, but they are also some of the hardest molecular features to analyze with confidence. Phosphorylation, asymmetric dimethylarginine (ADMA), and O-GlcNAcylation (O-GlcNAc) often occur at low abundance, can be highly labile, and frequently require precise site localization to understand their biological meaning. This technical note shows how the Thermo Scientific Orbitrap Excedion Pro Hybrid Mass Spectrometer helps address those challenges by combining fast, sensitive performance with complementary HCD and EThcD fragmentation workflows.

Thermo Fisher Scientific: Phosphorylation, ADMA, and O-GlcNAc Analysis on the Orbitrap Excedion Pro Mass Spectrometer

Using enriched PTM samples and automated data-dependent acquisition, the study demonstrates that the Orbitrap Excedion Pro MS can deliver both depth and confidence across multiple PTM classes. For phosphorylation analysis, the platform identified 5,892 unique phosphosites from IMAC-enriched samples and 2,941 unique phosphotyrosine sites after phosphotyrosine enrichment, highlighting strong performance for both broad and highly targeted signaling studies. Just as importantly, the data show that HCD and EThcD provide complementary value, with each fragmentation method contributing unique site identifications and helping resolve ambiguous assignments.

The note also highlights the value of EThcD for more analytically demanding PTMs. In ADMA-enriched samples, EThcD delivered more ADMA peptide-spectrum matches and more unique modified peptides than HCD, supporting improved confidence in site characterization. For O-glycopeptides, the Orbitrap Excedion Pro MS uses an intelligent HCD-product-dependent-EThcD strategy that triggers EThcD only when oxonium ions are detected, improving productivity while preserving the fragmentation detail needed for confident glycosite localization.

Overall, this technical note shows how Orbitrap Excedion Pro MS enables researchers to go deeper into PTM biology with greater sensitivity, better site confidence, and smarter acquisition. For scientists studying signaling, regulation, and disease mechanisms, it presents a powerful approach for expanding PTM coverage without compromising analytical rigor.

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

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