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Quantitative Analysis of Chromatin Proteomes in Disease
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Cleland immunoblotting quantifies cysteine proteoform ensembles.

James N Cobley1, Anna Noble2, Matthew Guille2

  • 1The University of Dundee, Dundee, Scotland, UK.

Free Radical Biology & Medicine
|April 16, 2026
PubMed
Summary

Cleland immunoblotting resolves cysteine proteoforms by encoding oxidation states. This new method quantifies protein oxidation ensembles, advancing proteoform research.

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Area of Science:

  • Biochemistry
  • Proteomics
  • Molecular Biology

Background:

  • Cysteine oxidation creates diverse proteoforms not detectable by standard methods.
  • Existing techniques collapse complex oxidation data into a single percentage.
  • The number of proteoforms increases exponentially with cysteine residues.

Purpose of the Study:

  • To develop a method for resolving and quantifying intact-protein oxidation-grade ensembles.
  • To introduce Cleland immunoblotting, a reversible mass-encoding strategy.
  • To enable the study of discrete cysteine proteoforms.

Main Methods:

  • Utilized the biotin-switch technique to label oxidized cysteines.
  • Employed a PEG reagent for oxidation-dependent mobility shifts.
  • Applied reductive cleavage with Cleland's reagent (DTT) for antibody accessibility.

Main Results:

  • Demonstrated resolution and quantification of distinct oxidation-grade ensembles.
  • Showcased binomial degeneracy in band structure, yielding distinguishable ensembles.
  • Successfully applied to cdc20 in Xenopus laevis oocytes.

Conclusions:

  • Cleland immunoblotting resolves and quantifies cysteine proteoform ensembles.
  • This technique advances the study of protein oxidation and proteoforms.
  • Enables detailed analysis of protein oxidation states.