Reducing offsite modifications using 2-mercaptoethanol for LC-MS analyses.

The alkylation of thiol groups in cysteine (Cys) residues is a critical step in liquid chromatography-mass spectrometry (LC-MS) analysis. However, conventional alkylation methods often introduce non-specific modifications at the N-terminal and other amino acids, thus complicating peptide identification and quantification using MS. To overcome this problem, we developed a novel method using 2-mercaptoethanol (2-ME) in the presence of dimethyl sulfoxide (DMSO) to specifically target Cys residues. Although 2-ME is typically used as a reducing agent for disulfide bonds, we demonstrated that DMSO promotes the specific binding of 2-ME to Cys in a concentration-dependent manner. Importantly, this approach significantly reduces offsite alkylation of other amino acids compared with conventional procedures, thereby improving peptide identification and enhancing the overall accuracy of quantification. This method offers a practical solution to the challenges posed by conventional alkylation techniques, provides more reliable MS data for peptides containing Cys residues, and enhances the accuracy of peptide quantification.