Cysteine alkylating methods in shotgun proteomics and their possible effects on methionine residues

Neatly every proteomic protocol has a Cysteine reduction and alkylation step in it preceding the trypsin digestion. It needs to be done in order to remove the s-s bonds and prevent proteins from folding, thus, provide better access for trypsin. Among various reagents used for alkylation, iodoacetamide (IAM) seems to be the most common. Although, it has been shown that IAM can cause some undesired side reactions [Chernobrovkin et al., 2015; Muller et al., 2017]. In this work, we have compared the effect of four alkylating agents: iodoacetamide (IAM), 4-vinylpyridine (4-VP), chloroacetamide (CAM) and S-Methyl methanethiosulfonate (MMTS). In addition, we tried to check whether post-treatment with DTT (p-red) is reasonable in the case of irreversible alkylation.