Fragment Smarter, See Deeper: How the Orbitrap Excedion Pro Unlocks EASY-ETD and HCD Fragmentation

When every peptide bond matters, fragmentation is where discovery begins. The Thermo Scientific Orbitrap Excedion Pro mass spectrometer brings EASY-ETD and HCD together on a single, ultra-high-performance Orbitrap platform—delivering clarity across proteomics, biopharma, and structural biology.

Two fragmentation technologies, one hybrid advantage

EASY-ETD — gentle by design, powerful by nature

EASY-ETD transfers electrons to peptide/protein ions, breaking the backbone while preserving labile PTMs and fragile linkages, generating c- and z-ions ideal for confident site localization.
Especially valuable for: phosphorylation, glycosylation, deamidation, isomer discrimination (Asp/IsoAsp; Leu/IsoLeu), and ADC linker–payload integrity.

Example — isomer discrimination with diagnostic ions
In a UHPLC-HRAM ddMS² peptide-mapping experiment on NISTmAb, EThcD (EASY-ETD + supplemental HCD) produced the ETD-diagnostic c+57 and z–57 ions and unambiguously identified isoAsp in a 23-aa, 3+ deamidated peptide present at 0.18%, with <8 ppm mass error for both signatures (Figure 1). This shows how electron-based fragmentation retains the information needed to resolve subtle isomerization that collision-only methods can miss.

Annotated EThcD MS² spectrum with c/z and b/y labels; insets show z6–57 and c17+57 peaks used to confirm isoAsp in a low-abundance deamidated peptide.

Figure 1: EASY-ETD/EThcD reveals isoAsp with signature ions.

EThcD full-MS² spectrum from a NISTmAb tryptic peptide-mapping run. Zoom-in panels highlight z6–57 and c17+57 confirming isoAsp at 0.18% with <8 ppm error.

HCD — High-Energy Confidence for every workflow

HCD generates rich b- and y-ion spectra for robust sequencing and quantitative reproducibility. With the Excedion Pro’s ultra-high-field Orbitrap (MS² up to 70 Hz) and—on the BioPharma edition—an extended mass range to m/z 12,000, HCD supports full sequence coverage, consistent DIA/DDA quantitation, and confident identification of oxidation, deamidation, and truncation variants. Together, EASY-ETD and HCD provide orthogonal fragmentation—comprehensive coverage and high-confidence confirmation in one analysis.

Why combine EASY-ETD and HCD?

PTM localization: EASY-ETD preserves fragile sites; HCD confirms sequence context.
ADC characterization: EASY-ETD keeps linker–payload intact; HCD supports quant and variant checks.
Isomer detection: EASY-ETD resolves Asp/IsoAsp; HCD adds complementary evidence.
Quant proteomics: EASY-ETD maintains structural integrity; HCD delivers reproducible intensity.
Throughput: Electron-driven fragmentation + fast collision MS² (up to 70 Hz).

Applications that push the limits

Biopharma Characterization

Map DARs and conjugation sites in ADCs with confidence
Detect low-level PTMs impacting efficacy
Combine native intact MS and EThcD mapping for complete characterization

EThcD MS² of ADYEK(188-MCC-DM1)HK with diagnostic c and z ions that localize the DM1 conjugation site at K188.

Figure 2. EThcD maps ADC conjugation without cleaving the payload.

EThcD MS² spectrum of ADYEK(188-MCC-DM1)HK (light chain, z=3). Colored arrows mark c and z fragments used to localize the conjugation site at K188; complementary y/c ions provide sequence context. Data acquired on the Orbitrap Excedion Pro BioPharma platform.

Advanced proteomics

Expand sequence coverage in complex proteomes with complementary ion types
Support Hybrid-DIA for accurate quantitation with fewer false positives
Profile labile PTMs that collision-only methods may miss

Structural biology & HDX-MS

Preserve H/D labeling and delicate interactions
Enable low-flow DIA-HDX with high sensitivity and throughput

Hardware that makes it possible

OptaMax Plus Ion Source — enhanced spray stability and ease of use
Advanced Quadrupole Technology (AQT) — ultra-selective precursor isolation
Ion-Routing Multipole (IRM) — HCD, ETD, and EThcD in a single module
Electrodynamic Ion Funnel (EDIF) — optimized transmission for labile compounds
Ultra-high-field Orbitrap analyzer — high mass accuracy and throughput; BioPharma edition supports m/z 12,000

Answering the “How” behind advanced fragmentation

How does EASY-ETD improve peptide analysis vs standard ETD?
Optimized ion routing with optional supplemental activation (EThcD) yields efficient electron transfer and improved sequence coverage—especially for labile or larger peptides.

Can HCD and EASY-ETD be used in one workflow?
Yes—EThcD provides complementary c/z and b/y information in a single MS² event, ideal for PTM localization and conjugation analysis.

What’s the benefit for biopharma labs?
More insight per sample. EASY-ETD localizes modifications or conjugation sites while HCD supports robust quantitation—accelerating analytical development and QC.

From complexity to confidence

The Orbitrap Excedion Pro lets scientists choose the right fragmentation energy for every molecule—uncovering fine-structure detail without sacrificing throughput. From intact ADCs to complex proteomes, EASY-ETD + HCD deliver the most complete view of molecular structure available on a hybrid Orbitrap platform.

Learn more about Orbitrap Excedion Pro Mass Spectrometers

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