Systematic Evaluation of Protein Reduction and Alkylation Reveals Massive Unspecific Side Effects by Iodine-containing Reagents
Systematic Evaluation of Protein Reduction and Alkylation Reveals Massive Unspecific Side Effects by Iodine-containing Reagents
复制标题
DOI：
10.1074/mcp.m116.064048
复制
发表时间：
2017-07-01
影响因子：
7
通讯作者：
Winter, Dominic
中科院分区：
文献类型：
--
作者：
Mueller, Torsten;Winter, Dominic
半胱氨酸残基的还原和烷基化是几乎任何蛋白质组学工作流程的一部分。尽管它经常被使用，但到目前为止，还没有系统地研究不同条件对蛋白质组学研究结果的影响。在这项研究中，我们比较了常用的还原试剂(二硫苏糖醇、三(2-羧乙基)膦和β-硫醇)和烷基化试剂(碘乙酰胺、碘乙酸、丙烯酰胺和氯乙酰胺)。使用凝胶内消化以及HeLa细胞胞浆部分的SAX分级溶液内消化，我们评估了13种不同的还原和烷基化条件，导致了相当不同的识别率。我们观察到7个氨基酸的异位烷基化反应以及在肽N末端的显著差异，鉴定了所有试剂的单加合物和双加合物。通过二甲基标记法、质量耐受性搜索和合成肽实验，我们确定含碘烷基化试剂对蛋氨酸残基的烷基化是造成差异的主要因素之一。我们观察到碘和非碘的烷基化试剂在凝胶内消化的样品中识别的含有蛋氨酸的多肽光谱匹配数量的差异超过9倍。这是由于氨甲基化和羧甲基化蛋氨酸侧链的形成，以及在ESI电离或MS/MS裂解过程中导致的显著中性损失，大大降低了含有蛋氨酸的多肽的识别率。用丙烯酰胺作为烷基化试剂效果最好，而用二硫苏糖醇和β-巯基乙醇分别还原溶液中和凝胶中消化的样品得到的肽谱匹配数最高。
Reduction and alkylation of cysteine residues is part of virtually any proteomics workflow. Despite its frequent use, up to date no systematic investigation of the impact of different conditions on the outcome of proteomics studies has been performed. In this study, we compared common reduction reagents (dithiothreitol, tris-(2-carboxyethyl)- phosphine, and beta-mercaptoethanol) and alkylation reagents (iodoacetamide, iodoacetic acid, acrylamide, and chloroacetamide). Using in-gel digests as well as SAX fractionated in-solution digests of cytosolic fractions of HeLa cells, we evaluated 13 different reduction and alkylation conditions resulting in considerably varying identification rates. We observed strong differences in offsite alkylation reactions at 7 amino acids as well as at the peptide N terminus, identifying single and double adducts of all reagents. Using dimethyl labeling, mass tolerant searches, and synthetic peptide experiments, we identified alkylation of methionine residues by iodine-containing alkylation reagents as one of the major factors for the differences. We observed differences of more than 9-fold in numbers of identified methionine-containing peptide spectral matches for in-gel digested samples between iodine-and noniodine-containing alkylation reagents. This was because of formation of carbamidomethylated and carboxymethylated methionine side chains and a resulting prominent neutral loss during ESI ionization or in MS/MS fragmentation, strongly decreasing identification rates of methionine-containing peptides. We achieved best results with acrylamide as alkylation reagent, whereas the highest numbers of peptide spectral matches were obtained when reducing with dithiothreitol and beta-mercaptoethanol for the in-solution and the in-gel digested samples, respectively.