Cysteine oxidation generates biologically important proteoforms that are not directly resolvable using conventional immunoblot or peptide-based mass spectrometry workflows. For a protein containing R cysteine residues, the number of theoretical proteoforms expands exponentially ( 2ˆR ), while aggregate oxidation measurements collapse this space into a single percentage value. Here, we present Cleland immunoblotting, a reversible mass-encoding strategy that resolves and quantifies intact-protein oxidation-grade ensembles defined by oxidation integer. Each band contains co-migrating cysteine proteoforms with the same number of oxidised cysteines, and the degeneracy of each band follows the binomial theorem. Using the biotin-switch technique, reversibly oxidised cysteines are labelled with a 2-pyridyldithiol-functionalised polyethylene glycol (PEG) reagent to encode oxidation-dependent mobility shifts during gel electrophoresis. Following electrophoretic separation, the PEG linkage is reductively removed in-gel using Cleland’s reagent (DTT) prior to membrane transfer, restoring antibody accessibility while preserving band resolution. Band structure follows binomial degeneracy, yielding r + 1 oxidation-graded ensembles that are experimentally distinguishable. Application to cdc20 in Xenopus laevis oocytes demonstrates resolution and quantification of discrete fully reduced and oxidised cysteine protoeforms. By resolving cysteine proteoform ensembles, Cleland immunoblotting stands to advance proteoform research. • Cleland immunoblotting resolves intact cysteine oxidation-grade bands • Reversible PEG tagging preserves mobility shifts but restores antibody binding • In-gel DTT removes 2PB before transfer, overcoming PEG-induced epitope masking • Band structure follows oxidation integer and binomial degeneracy • Binary cdc20 cysteine proteoforms were quantified during Xenopus fertilisation
Cobley et al. (Wed,) studied this question.