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首页> 外文期刊>Biochimica et biophysica acta: BBA: International journal of biochemistry, biophysics and molecular biololgy. Proteins and Proteomics >Characterization of reaction conditions providing rapid and specific cysteine alkylation for peptide-based mass spectrometry
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Characterization of reaction conditions providing rapid and specific cysteine alkylation for peptide-based mass spectrometry

机译：反应条件的表征为基于肽的质谱分析提供了快速而特异性的半胱氨酸烷基化

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Alkylation converts Cys thiols to thioethers and prevents unwanted side reactions, thus facilitating mass spectrometric identification of Cys-containing peptides. Alkylation occurs preferentially at Cys due to its high nucleophilicity, however reactions at other such sites are possible. N-ethylmaleimide (NEM) shows rapid reaction kinetics with Cys and careful definition of reaction conditions results in little reactivity at other sites. Analysis of a protein standard alkylated under differing reaction conditions (pH, NEM concentrations and reaction times) was performed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and selected reaction monitoring (SRM) of NEM-modified and unmodified peptide pairs. Mis-alkylation sites at primary and secondary amines were identified and limited to one equivalent of NEM. No evidence for hydroxyl or thioether alkylation was observed. Improved specificity was achieved by restricting the pH below neutral, NEM concentration below 10 mM and/or reaction time to below 5 min. Maximal removal of Cys activity was observed in tissue homogenates at 40 mM NEM within 1 min, dependent upon efficient protein denaturation. SRM assays identified peptide-specific levels of mis-alkylation, indicating that NEM-modified to unmodified ratios did not exceed 10%, with the exception of Cys alkylation that proceeded to 100%, and some Lys residues that resulted in tryptic missed cleavages. High reactivity was observed for His residues considering their relatively low abundance. These data indicate that rapid and specific Cys alkylation is possible with NEM under relatively mild conditions, with more abrasive conditions leading to increased non-specific alkylation without appreciable benefit for MS-based proteomics. ? 2012 Elsevier B.V. All rights reserved.
机译：烷基化将Cys巯基转化为硫醚并防止了不良的副反应，从而促进了质谱鉴定含Cys的肽。由于其高亲核性，烷基化优先发生在Cys上，但是在其他此类位点也可能发生反应。 N-乙基马来酰亚胺（NEM）显示与Cys的快速反应动力学，对反应条件的仔细定义导致在其他位点的反应性很小。使用液相色谱-串联质谱（LC-MS / MS）和NEM修饰和未修饰肽的选定反应监测（SRM）进行在不同反应条件（pH，NEM浓度和反应时间）下烷基化的蛋白质标准品的分析对。确定了伯胺和仲胺的误烷基化位点，并将其限制为一当量的NEM。没有观察到羟基或硫醚烷基化的证据。通过将pH值限制在中性以下，将NEM浓度限制在10 mM以下和/或将反应时间限制在5分钟以下，可以提高特异性。在1分钟内，在40 mM NEM的组织匀浆中观察到Cys活性的最大去除，这取决于有效的蛋白质变性。 SRM分析确定了肽特异性的错误烷基化水平，表明NEM修饰至未修饰的比例不超过10％，Cys烷基化进行至100％除外，并且某些Lys残基导致胰蛋白酶错过了裂解。考虑到他的残留物相对较低的丰度，观察到高反应性。这些数据表明，NEM可以在相对温和的条件下进行快速而特异性的Cys烷基化，更多的研磨条件导致非特异性烷基化的增加，而对基于MS的蛋白质组学没有明显的好处。 ？ 2012 Elsevier B.V.保留所有权利。

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