Quantification of Site-Specific Disulfide Bond Redox States in Proteins by Parallel Reaction Monitoring-Mass Spectrometry (PRM-MS).

Conventional non-targeted approaches using data-dependent acquisition (DDA) with isotopic labeling mass spectrometry (MS) have demonstrated limited effectiveness in characterizing site-specific disulfide bond redox states, primarily due to suboptimal coverage and inconsistent reproducibility. Here, we introduce a targeted approach employing differential cysteine alkylation coupled with parallel reaction monitoring (PRM)-MS to identify the redox states of specific disulfide bond sites in proteins, which significantly improves the coverage and reproducibility of mass spectrometry data.