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2-Mercaptoethanol/DMSO Workflow Enables Highly Reproducible Quantitative Proteomics
Arisa Suto1, Yoshihiro Ishikawa1, Toshihide Matsumoto2
1Department of Physics, School of Science, Kitasato University, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0373, Japan.
Journal of Proteome Research
|May 8, 2026
Summary
A new 2-mercaptoethanol (2-ME)/dimethyl sulfoxide (DMSO) workflow improves quantitative proteomics accuracy and reproducibility. This method enhances cysteine modification detection and peptide coverage, crucial for understanding cellular functions and disease mechanisms.
Area of Science:
- Proteomics
- Mass Spectrometry
- Biochemistry
Background:
- Proteomics offers insights into cellular functions and disease mechanisms.
- Conventional iodoacetamide (IAA) carbamidomethylation for cysteine alkylation introduces nonspecific modifications, increasing spectral complexity and reducing quantitative accuracy in mass spectrometry.
- There is a need for improved, reproducible proteomics workflows for accurate quantitative analysis.
Purpose of the Study:
- To establish and evaluate a reproducibility-focused 2-mercaptoethanol (2-ME)/dimethyl sulfoxide (DMSO) workflow for quantitative proteomics.
- To systematically assess the quantitative performance of the 2-ME/DMSO workflow at the proteome-wide level compared to conventional IAA treatment.
- To demonstrate the utility of the new workflow in detecting biologically relevant alterations in disease models.
Main Methods:
- Mouse liver proteomes were processed using either the optimized 2-ME/DMSO workflow or conventional IAA treatment.
- Samples were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
- Quantitative performance was evaluated based on the number of cysteine-modified peptides, protein sequence coverage, and quantitative reproducibility (coefficient of variation, CV).
Main Results:
- The optimized 2-ME treatment increased the identification of cysteine-modified peptides by 1.6- to 1.9-fold.
- While total protein identifications remained comparable, 77% of proteins showed improved sequence coverage with the 2-ME/DMSO workflow.
- Quantitative reproducibility significantly improved: peptide CV ≤ 20% increased from 61.4% (IAA) to 86.1% (2-ME/DMSO), and protein CV ≤ 20% increased from 80.6% (IAA) to 93.5% (2-ME/DMSO).
- The workflow successfully detected cisplatin-induced alterations in ovarian clear cell carcinoma.
Conclusions:
- The 2-ME/DMSO workflow provides a simple, highly reproducible strategy for accurate quantitative proteomics.
- This method enhances cysteine modification detection and improves quantitative accuracy, enabling more reliable insights into biological systems.
- The workflow is suitable for various applications, including disease mechanism studies and biomarker discovery.
