A Scalable Low-Input Redox Proteomics Workflow Enables High-Throughput Analysis of Cysteine Oxidation in 96-Well Cell Cultures.

Reversible oxidation of cysteine residues (redox modifications) plays a crucial role in regulating protein function, signaling, and cellular homeostasis. These dynamic modifications act as molecular switches that transduce redox signals and modulate stress responses, metabolism, and pathogenesis. Redox proteomics enables systematic profiling of these modifications, quantifying the oxidation levels of tens of thousands of cysteine sites across the proteome and providing rich data to understand redox-regulated networks. However, conventional redox proteomic workflows are often limited by low-throughput and high sample requirements. Here, we present a high-throughput sample processing workflow for redox proteomics analysis from as little as 2 μg of protein. The workflow integrates 96-well plate-based cell culture, lysis, digestion, and cysteine-peptide enrichment, substantially increasing throughput and reducing hands-on processing time. Incorporating field asymmetric ion mobility spectrometry (FAIMS) further enhances redox proteome coverage by removing singly charged species in low-input samples, thereby increasing the signal of cysteine-containing peptides. Applying the workflow to RAW264.7 cells cultured in 96-well plates (40,000 cells per well), DIA identified >10,000 cysteine sites, and revealed a global increase in cysteine oxidation upon diamide treatment. To assess robustness, we repeated the 96-well experiment across three independent batches processed on different days and observed consistent coverage, reproducible quantification, and comparable diamide-induced oxidation of heat shock proteins, transcription factors, and protein kinases. Together, this workflow and new data acquisition scheme enable comprehensive redox proteomics from minimal inputs, paving the way for high-throughput sophisticated studies of redox modifications in cell signaling, disease, or large-scale screening of redox-modulating compounds.