Cysteine alkylating methods in shotgun proteomics and their possible effects on methionine residues

Neatly every proteomic protocol has a Cysteine reduction and alkylation step in it preceding the trypsin digestion. It needs to be done in order to remove the s-s bonds and prevent proteins from folding, thus, provide better access for trypsin. Among various reagents used for alkylation, iodoacetamide (IAM) seems to be the most common. Although, it has been shown that IAM can cause some undesired side reactions [Chernobrovkin et al., 2015; Muller et al., 2017]. In this work, we have compared the effect of four alkylating agents: iodoacetamide (IAM), 4-vinylpyridine (4-VP), chloroacetamide (CAM) and S-Methyl methanethiosulfonate (MMTS). In addition, we tried to check whether post-treatment with DTT (p-red) is reasonable in the case of irreversible alkylation.

几乎每一项蛋白质组学实验方案均会在胰蛋白酶消化前设置半胱氨酸还原与烷基化（Cysteine reduction and alkylation）步骤。该步骤的作用在于断裂蛋白质的二硫键、防止分子折叠，从而提升胰蛋白酶的酶切可及性。在诸多用于烷基化的试剂中，碘乙酰胺（iodoacetamide，IAM）是最为常用的一种。不过已有研究表明，IAM可引发若干非预期副反应[Chernobrovkin等，2015；Muller等，2017]。本研究对比了四种烷基化试剂的应用效果：碘乙酰胺（IAM）、4-乙烯基吡啶（4-vinylpyridine，4-VP）、氯乙酰胺（chloroacetamide，CAM）以及S-甲基甲烷硫代磺酸酯（S-Methyl methanethiosulfonate，MMTS）。此外，我们还尝试探究在不可逆烷基化场景下，采用二硫苏糖醇（DTT）进行后处理（p-red）是否具有合理性。