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Study · Not a test of the peptide alone · 2025

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Electron Capture Dissociation for Discovery Top-Down Proteomics of Peptides and Small Proteins on Chromatographic Time Scales.

Manly LS, Roberts AM, Beckman JS, Roberts BR

Journal of the American Society for Mass Spectrometry28 Aug 2025Cited by 6 (Europe PMC)

PMID40877745 (opens in a new tab)PMCIDPMC12492394 (opens in a new tab)DOI10.1021/jasms.5c00116 (opens in a new tab)PepBankPUB-40877745
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Summary

Bottom-up proteomics introduces proteoform ambiguity due to the loss of connectivity between peptides and their original proteoforms. Top-down proteomics (TDP) removes the ambiguity through the direct identification and characterization of intact proteoforms and their respective post-translational modifications (PTM). Electron capture dissociation (ECD) is an efficient and gentle peptide and protein fragmentation strategy that can be used for both bottom-up and top-down approaches. Here, we used an Agilent 6550 Q-TOF mass spectrometer retrofitted with an e-MSion ECD cell. Top-down sequencing capabilities of the cell were evaluated by sequencing of intact peptides and proteins on high-performance liquid chromatography (HPLC) time scales. Amyloid beta 1-40 (Aβ40) was first tested due to its pathophysiological role in Alzheimer's disease and served as our large peptide standard. We sequenced Aβ40 via reverse-phase HPLC-MS and achieved 95% sequence coverage on chromatographic time scales utilizing a data-dependent acquisition (DDA)-based method. Acetone-precipitated protein extracts from human brain were then separated by HPLC and analyzed with a DDA method, which identified 16 proteoforms between 2 and 17 kDa with sequence coverage ranging from 7 to 90% based on proteoform size and composition. In addition to proteoform identification, ECD fragmentation distinguished multiple isoaspartate modifications from aspartate, as well as accurately differentiating leucine from isoleucine residues directly from the human brain sample. Here, we observed isoaspartate within a thymosin beta-4 proteoform. Additionally, we demonstrated the differentiation of leucine and isoleucine within a subunit of ubiquitin. This study advances the application of LC-Q-TOF instrumentation for discovery-based top-down proteomics utilizing ECD as enabled by the e-MSion ECD cell.
CC BYText from Europe PMC. Highlighted sentences are the source of results.

Results from this study1

No result from this study comes from a test of the peptide.

Studies that did not test the peptide alone1

In this study, Thymosin beta-4 (human) was a lab tool or a carrier, part of a mix, or the subject of a lab method. These results are not evidence about taking Thymosin beta-4 (human).

  1. 01LabNot a test of Thymosin beta-4 (human) aloneEx vivo tissueOther

    Electron capture dissociation top-down proteomics of human brain protein extract detected isoaspartate within a thymosin beta‑4 proteoform.

    In plain wordsIn a lab method test on human brain extract, scientists found a chemically altered form of thymosin beta‑4.

    Peptide
    Thymosin beta-4 (human)
    Outcome
    Not applicableIsoaspartate in a thymosin beta-4 proteoform
    Study

    humann: not reported in source

    acetone-precipitated protein extract of human brain
    Outcome group
    Analytical methods and detection · Nervous system and cognition

    Source sentences

    Summary
    Here, we observed isoaspartate within a thymosin beta-4 proteoform.
    1 context sentence
    Summary · context
    Acetone-precipitated protein extracts from human brain were then separated by HPLC and analyzed with a DDA method, which identified 16 proteoforms between 2 and 17 kDa with sequence coverage ranging from 7 to 90% based on proteoform size and composition.

    In this study, Thymosin beta-4 (human) was a lab tool or a carrier, part of a mix, or the subject of a lab method. This result is not evidence about taking Thymosin beta-4 (human).

AI wrote and checked these results. AI can make mistakes, so check important results against the source.

Version history

PepBank keeps every version of the data on this study. Status changes from Retraction Watch, Europe PMC and Crossref are listed with their source.

  1. Version 2Current

    13 Oct 2026, 01:52 UTC

    Study results updated

  2. Version 1

    12 Oct 2026, 18:17 UTC

    First added · source update from Europe PMC

Subjects

Terms as the source lists them.

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