The Impact of Commonly Used Alkylating Agents on Artifactual Peptide Modification
The Impact of Commonly Used Alkylating Agents on Artifactual Peptide Modification
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DOI：
10.1021/acs.jproteome.7b00022
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发表时间：
2017-09-01
影响因子：
4.4
通讯作者：
Robinson, Phillip J.
中科院分区：
文献类型：
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作者：
Hains, Peter G.;Robinson, Phillip J.
碘乙酰胺是迄今为止在蛋白质组学样品制备过程中最常用的半胱氨酸烷基化剂。另一种选择，2-氯乙酰胺，最近被建议减少半胱氨酸以外的残基的烷基化，如n端，Asp, Glu, Lys, Ser， Thr和Tyr。在这里，我们表明，虽然2-氯乙酰胺降低了脱靶烷基化的水平，但它表现出一系列的不利影响。其中最显著的是蛋氨酸氧化，在所有含met的肽中，蛋氨酸氧化最多增加到40%，而在碘乙酰胺中，这一比例为2-5%。也观察到单和二氧化色氨酸的增加。在其他一系列翻译后修饰和消化参数方面，没有观察到烷基化试剂之间的其他差异。对来自三个不同供应商的2-氯乙酰胺进行了有害影响观察。2-氯乙酰胺对蛋氨酸氧化的不利影响表明它不是理想的蛋白质组学烷基化试剂。
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.