ProCan and the Cell Signalling Units, Children's Medical Research Institute, The University of Sydney , 214 Hawkesbury Road, Westmead, NSW 2145, Australia.
J Proteome Res. 2017 Sep 1;16(9):3443-3447. doi: 10.1021/acs.jproteome.7b00022. Epub 2017 Aug 23.
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.
碘乙酰胺是迄今为止用于蛋白质组学样品制备过程中半胱氨酸烷基化的最常用试剂。最近有人提出，替代试剂 2-氯乙酰胺可以减少除半胱氨酸以外的残基（如 N 末端、天冬氨酸、谷氨酸、赖氨酸、丝氨酸、苏氨酸和酪氨酸）的烷基化。在这里，我们发现尽管 2-氯乙酰胺降低了非靶向烷基化的水平，但它表现出一系列的不良反应。其中最显著的是蛋氨酸氧化，与碘乙酰胺相比，所有含有蛋氨酸的肽段中蛋氨酸氧化增加到最大值的 40%，而碘乙酰胺为 2-5%。单氧化和二氧化色氨酸也有增加。在一系列其他翻译后修饰和消化参数方面，没有观察到两种烷基化试剂之间的其他差异。来自三个不同供应商的 2-氯乙酰胺都观察到了这种有害影响。2-氯乙酰胺对蛋氨酸氧化的不利影响表明，它不是蛋白质组学的理想烷基化试剂。
