5 years ago

The Impact of Commonly Used Alkylating Agents on Artifactual Peptide Modification

The Impact of Commonly Used Alkylating Agents on Artifactual Peptide Modification
Peter G. Hains, Phillip J. Robinson
Iodoacetamide is by far the most commonly used agent for alkylation of cysteine during sample preparation for proteomics. An alternative, 2-chloroacetamide, has recently been suggested to reduce the alkylation of residues other than cysteine, such as the N-terminus, Asp, Glu, Lys, Ser, Thr, and Tyr. Here we show that although 2-chloroacetamide reduces the level of off-target alkylation, it exhibits a range of adverse effects. The most significant of these is methionine oxidation, which increases to a maximum of 40% of all Met-containing peptides, compared with 2–5% with iodoacetamide. Increases were also observed for mono- and dioxidized tryptophan. No additional differences between the alkylating reagents were observed for a range of other post-translational modifications and digestion parameters. The deleterious effects were observed for 2-chloroacetamide from three separate suppliers. The adverse impact of 2-chloroacetamide on methionine oxidation suggests that it is not the ideal alkylating reagent for proteomics.

Publisher URL: http://dx.doi.org/10.1021/acs.jproteome.7b00022

DOI: 10.1021/acs.jproteome.7b00022

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