2-Mercaptoethanol/DMSO workflow enables highly reproducible quantitative proteomics

Proteomics provides a systematic and high-throughput approach to comprehensively characterize protein networks, enabling insights into cellular functions and disease mechanisms. Carbamidomethylation using iodoacetamide (IAA), a common method for cysteine alkylation, is known to cause nonspecific modifications that increase MS spectral complexity and reduce quantitative accuracy. Here, we extended our 2-mercaptoethanol (2-ME) adduction method, enhanced by dimethyl sulfoxide (DMSO), from single-protein applications to a proteome-wide workflow. Mouse liver proteomes were processed using either 2-ME/DMSO or conventional IAA treatment, followed by LC-MS/MS analysis. The optimized 2-ME treatment increased the number of cysteine-modified peptides by 1.6-1.9-fold. Although total protein identifications were comparable, 77% of proteins exhibited improved sequence coverage. Quantitative reproducibility was also enhanced, with peptides quantified coefficient of variation (CV) [≤] 20% increasing from 61.5% (IAA) to 86.1% (2-ME), and proteins from 80.6% to 93.5%. Application of the workflow to an ovarian clear cell carcinoma reliably detected cisplatin-induced alterations. The 2-ME/DMSO workflow offers a simple and highly reproducible proteomics strategy for accurate quantitative proteomics.