pr7b00022_si_002.xls (28 kB)
The Impact of Commonly Used Alkylating Agents on Artifactual Peptide Modification
dataset
posted on 2017-08-11, 00:00 authored by Peter G. Hains, Phillip J. RobinsonIodoacetamide 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.