PTM Analysis using HCD and EThcD on an Orbitrap Excedion Pro Mass Spectrometer

Significance of the topic

The confident detection and localization of post-translational modifications (PTMs) is essential for understanding cell signaling, protein function and disease mechanisms. PTMs are typically low-abundance, dynamic and labile during fragmentation, so analytical workflows must combine sensitive enrichment, rapid and high-resolution LC-MS acquisition, and fragmentation strategies that preserve modification information. Comparing collision-induced (HCD) and electron-transfer-based (EThcD) fragmentation on a modern Orbitrap platform clarifies trade-offs in identification depth, sequence coverage and site localization for diverse PTMs including phosphorylation, phosphotyrosine, asymmetric dimethylarginine (ADMA) and O-linked glycosylation.

Objectives and study overview

This study evaluates data-dependent acquisition (DDA) workflows using HCD and EThcD on the Thermo Scientific Orbitrap Excedion Pro mass spectrometer for identification and site localization of enriched PTM peptide samples. The aims were to (1) assess sensitivity and speed for enriched PTM samples with wide dynamic range, (2) compare identification counts and site assignment confidence between HCD and EThcD, and (3) demonstrate practical benefits of automatic per-precursor optimization of collision energy and ETD reaction settings in routine DDA analyses.

Methodology and instrumentation

Sample preparation and enrichment:

• Cell and tissue sources: Human HCT116 colon cells (trypsin-digested) and mouse liver and brain samples (trypsin or LysC digestion).
• Enrichment strategies: IMAC for global phosphopeptides; PTMScan kits (Cell Signaling Technology) for phosphotyrosine, ADMA and O-GlcNAc/O-glycosylation captures.

LC-MS acquisition and fragmentation:

• LC system: Thermo Vanquish Neo UHPLC with IonOpticks Aurora Ultimate 25 × 75 µm XT C18 column.
• Mass spectrometer: Thermo Scientific Orbitrap Excedion Pro with ETD capability.
• Acquisition: Data-dependent acquisition (DDA) using either HCD or EThcD for MS2 fragmentation. Collision energies and ETD reaction times were automatically optimized per precursor in the method.

Data processing:

• PTM-aware search workflows were applied to localize modifications; MSFragger and other PTM search tools are referenced as commonly used components in such analyses.

Main results and discussion

Overall performance:

• The Orbitrap Excedion Pro platform provided high mass accuracy, sensitivity and acquisition speed suitable for enriched PTM samples that contain many low-abundance modified peptides.
• Automatic optimization of collision energy and ETD reaction parameters simplified method setup while maintaining high-quality MS2 spectra across a wide precursor population.

Phosphorylation (global IMAC enrichment):

• Combined HCD and EThcD analyses yielded over 5,890 unique phosphosites. In that dataset phosphoserine dominated (~92% of sites); phosphotyrosine was relatively rare in the global phosphopeptide population reported in that experiment.

Phosphotyrosine enrichment (PTMScan pY kit):

• Targeted enrichment followed by DDA produced more than 2,900 unique phosphotyrosine sites. Both HCD and EThcD provided many confident peptide identifications and site localizations, with a portion of sites detected exclusively by one fragmentation mode.
• Summary table comparisons showed HCD identifying a larger number of proteins/peptides in the pY-enriched set (for example, higher total peptide and PSM counts), while EThcD still delivered strong phosphopeptide identification and complementary coverage (fewer total IDs but many unique site-localizing spectra).
• Example peptides demonstrated the practical value of EThcD: where HCD lacked sufficient fragment coverage in N-terminal regions for unambiguous site assignment, EThcD spectra often supplied the needed c/z-type ions to localize the modification confidently.

ADMA (asymmetric dimethylarginine) enrichment:

• Enrichment for ADMA-modified peptides yielded nearly 300 ADMA sites and comprised approximately 10% of identified peptides in the enriched samples.
• Although HCD produced more total peptide identifications in these datasets, EThcD produced more ADMA peptide-spectrum matches (PSMs) and a greater number of unique ADMA site identifications, indicating improved sensitivity for this modification class when using ETD-centered fragmentation.

O-linked glycosylation (O-GlcNAc and O-glycans):

• O-linked glycopeptides are labile under collision-based dissociation, complicating sequence and site assignment. EThcD—especially when triggered by oxonium ions—greatly improved detection of peptide backbone fragments that retain site information and thus increased confident localization of O-glycosylation sites.
• A concrete example showed stepHCD providing good sequence coverage but mis-assigning HexNAc positions, while EThcD produced richer fragment ion series enabling correct assignment of two HexNAc sites on a PCLO peptide.

Benefits and practical applications

• EThcD complements HCD by reducing neutral loss and supplying complementary fragment ion types (c/z ions), improving site localization for labile and multiply modified peptides.
• The Orbitrap Excedion Pro platform combines high resolution/accuracy with ETD capability and automatic optimization of fragmentation parameters, enabling robust DDA workflows for PTM-enriched samples in discovery proteomics and targeted PTM mapping.
• Laboratories performing phosphoproteomics, ADMA profiling, or glycoproteomics can benefit from integrating EThcD into routine acquisition to increase confidence in modification site assignments and to recover identifications missed by collision-only methods.

Future trends and possibilities

• Hybrid fragmentation strategies and intelligent triggering (e.g., oxonium-ion-triggered EThcD) will continue to improve glycopeptide detection and localization, and are likely to be integrated into automated methods for broader PTM classes.
• Advances in real-time method optimization and acquisition (per-precursor tuning) combined with improved search algorithms for open and PTM-centric searches will increase depth and confidence of PTM maps from limited sample material.
• Combining complementary fragmentation (HCD, EThcD, stepped collision energies) and data analysis tools that jointly interpret multiple fragmentation types will provide more complete sequence coverage and more reliable site localization for complex proteoforms.

Conclusions

• DDA methods on the Orbitrap Excedion Pro deliver fast, sensitive and high-confidence PTM analyses. Both HCD and EThcD produce large numbers of PTM identifications, with each mode adding unique and complementary identifications.
• EThcD is particularly valuable for labile modifications (O-glycosylation) and for cases where improved c/z fragment coverage enhances site localization (phosphotyrosine and ADMA). Automatic per-precursor optimization simplifies acquisition while preserving spectral quality across diverse PTM-enriched samples.

Instrumentation

• Vanquish Neo UHPLC system (Thermo Fisher Scientific).
• IonOpticks Aurora Ultimate 25 × 75 µm XT C18 UHPLC column.
• Orbitrap Excedion Pro mass spectrometer with ETD capability (Thermo Scientific).
• Sample enrichment: IMAC and PTMScan kits (Cell Signaling Technology).

Reference

1. Riley N, Malaker S, Driessen M, Bertozzi C. Journal of Proteome Research. 2020;19(8):3286–3301.