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 spectral complexity in mass spectrometry and reduce quantitative accuracy. Here, we established a reproducibility-focused 2-mercaptoethanol (2-ME)/dimethyl sulfoxide (DMSO) workflow and systematically evaluated its quantitative performance at the proteome-wide level. Mouse liver proteomes were processed using either 2-ME/DMSO or conventional IAA treatment, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The optimized 2-ME treatment increased the number of cysteine-modified peptides by 1.6- to 1.9-fold. Although total protein identifications were comparable, 77% of proteins exhibited improved sequence coverage with the optimized 2-ME treatment. Quantitative reproducibility was also enhanced, with the peptide quantified CV ≤ 20% increasing from 61.4% with IAA treatment to 86.1% with 2-ME treatment, and protein quantified CV ≤ 20% increasing from 80.6% with IAA treatment to 93.5% with 2-ME treatment. Application of this new workflow to 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.