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Protein tandem mass spectrometry (MS/MS) often generates sequence-informative fragments from backbone bond cleavages near the termini. This lack of fragmentation in the protein interior is particularly apparent in native top-down MS. Improved sequence coverage, critical for reliable annotation of posttranslational modifications (PTMs) and sequence variants, may be obtained from internal fragments generated by multiple backbone cleavage events. However, internal fragment assignments can be error prone due to isomeric/isobaric fragments from different parts of a protein sequence. Also, internal fragment generation propensity depends on the chosen MS/MS activation strategy. Here, we examine internal fragment formation in electron capture dissociation (ECD) and electron transfer dissociation (ETD) following native and denaturing MS, as well as liquid chromatography (LC)/MS of several proteins. Experiments were undertaken on multiple instruments, including Q-ToF, Orbitrap, and high-field FT-ICR across four laboratories. ECD was performed at both ultrahigh vacuum and at similar pressure to ETD conditions. Two complementary software packages were used for data analysis. When feasible, ETD-higher-energy collision dissociation (ETD-HCD) MS3 was performed to validate/refute potential internal fragment assignments, including differentiating MS3 fragmentation behavior of radical vs. even-electron primary fragments. We show that, under typical operating conditions, internal fragments cannot be confidently assigned in ECD, nor ETD. On the other hand, such fragments, along with some b-type terminal fragments (not typically observed in ECD/ETD spectra) appear at atypical ECD operating conditions, suggesting they originate from a separate ion-electron activation process. Furthermore, atypical fragment ion types, e.g., x ions, are observed at such conditions as well as upon EThcD, presumably due to vibrational activation of radical z-type ions.
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This content will become publicly available on March 1, 2026
Extraction anode lens effects in gas phase peptide cation-electron reactions
Gas phase cation-electron reactions, from electron capture dissociation (ECD; <1 eV electrons) to electron ionization dissociation (>~26 eV electrons), are highly beneficial for biomolecular structural characterization. These techniques offer high sequence coverage, labile posttranslational modification retention, and sidechain loss fragments which can differentiate isomeric residues. For optimum performance, careful tuning of electron energy, flux, and irradiation time is required to reach efficiency at a particular energy regime. The cathode bias voltage (CBV) is the primary determinant of electron energy, while several parameters including CBV, extraction anode lens voltage (LV), and cathode heating current determine electron flux. We present an in-depth examination of how the interplay of these parameters at variable irradiation times results in differing peptide cation-electron reaction regimes. A particularly interesting finding was the prominent high energy fragmentation pathways observed at low (~- 1.0 V) CBV and high (>50 V) LV, as compared with conventional (~5 V) LV for peptide ECD. Specifically, high LV resulted in tandem ionization, observed for both singly- and doubly protonated peptides, alongside increased sequence coverage for both charge states from complex spectra containing a multitude of a/b/c/d/w/x/y/z•-type terminal fragments as well as internal fragments and a large number of neutral losses. Electron flux and energy measurements as well as electron irradiation at constant flux showed that an increased number of higher energy electrons are present at high vs. low LV, i.e., the observed “lens effect” is likely due to the presence of high energy electrons under such conditions. This extraction anode lens effect may explain previous observations of unexpected internal fragments from ECD.
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- PAR ID:
- 10610167
- Publisher / Repository:
- Elsevier
- Date Published:
- Journal Name:
- International Journal of Mass Spectrometry
- Volume:
- 509
- Issue:
- C
- ISSN:
- 1387-3806
- Page Range / eLocation ID:
- 117390
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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