The impact of commonly used alkylating agents on artefactual peptide modification

ABSTRACT: Iodoacetamide is by far the most commonly used agent for alkylation of cysteine during sample preparation for proteomics. An alternative, 2-chloroacetamide, has been recently 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 was methionine oxidation, which increases to a maximum of 40% of all Met containing peptides compared to 2-5% with iodoacetamide. Increases were also observed for mono and dioxidised tryptophan. No additional differences between the alkylating reagents were observed for a range of other post-translational modifications and digestion parameters. The adverse impact of 2-chloroacetamide on methionine oxidation suggest it is not the ideal alkylating reagent for proteomics.

INSTRUMENT(S): TripleTOF 6600

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Testis

SUBMITTER: Peter Hains  

LAB HEAD: Philip J Robinson

PROVIDER: PXD007071 | Pride | 2017-08-16

REPOSITORIES: Pride