Cleland immunoblotting quantifies cysteine proteoform ensembles

Cysteine oxidation generates biologically important proteoforms that are difficult to directly resolve using conventional immunoblot or peptide-based mass spectrometry workflows. For a protein containing R cysteine residues, the number of theoretical cysteine proteoforms expands exponentially (2^R), 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 cysteine proteoform ensembles by their 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-payload 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 can advance proteoform research.