Optimised two-dimensional electrophoresis procedures for the protein characterisation of structural tissues
Optimised two-dimensional electrophoresis procedures for the protein characterisation of structural tissues
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DOI：
10.1002/pmic.200401073
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发表时间：
2005-05-01
期刊：
影响因子：
3.4
通讯作者：
Tighe, PJ
中科院分区：
文献类型：
--
作者：
Hopkinson, A;McIntosh, RS;Tighe, PJ
结构组织的蛋白质分析通常是非常有问题的。羊膜具有独特的伤口愈合和抗瘢痕形成特性，然而，关于其活性蛋白含量知之甚少。羊膜的结构性质需要整个二维（2-D）工作流程的开发和广泛优化。使用强力增溶缓冲液提取蛋白质，并使用2-D电泳进行分析，然后进行质谱（MS）鉴定。保存和加工导致可溶性结构和膜相关蛋白质的预分级。通过使用N，N-二甲基丙烯酰胺（DMA）烷基化和双（2-羟乙基）二硫化物（HED）的半胱氨酸封闭实现了增强的蛋白质溶解度;证明了HE D的有效应用的替代程序。还评估了沉淀和杯加载与凝胶内再水化的益处，并描述了HED与后者的雇用程序。优化后，使用MS从羊膜中鉴定出代表性样品21种蛋白质，验证程序与MS相容。我们的结果表明，从蛋白质结构组织中分离蛋白质的技术已经得到了优化。简言之，使用含有载体两性电解质、二硫苏糖醇（DTT）和3-（环己基氨基）-1-丙磺酸（CHAPS）的硫脲/尿素提取缓冲液提取蛋白质。DMA烷基化后，沉淀蛋白质（使用来自阿默舍姆Biosciences的2-D清除试剂盒）并在含有较低浓度DTT的提取缓冲液中重新溶解。将样品杯加载到再水化的含HED的条带上或再缓冲到含HED的缓冲液中，然后进行凝胶内再水化。
The protein analysis of structural tissues is typically highly problematic. Amniotic membrane displays unique wound healing and anti-scarring properties; however, little is known concerning its active protein content. The structural nature of amniotic membrane necessitated development and extensive optimisation of the entire two-dimensional (2-D) workflow. Proteins were extracted using powerful solubilisation buffers and analysis carried out using 2-D electrophoresis followed by mass spectrometry (MS) identification. Preservation and processing resulted in pre-fractionation of soluble from structural and membrane-associated proteins. Enhanced protein solubility was achieved by cysteine blocking using both N,N-dimethylacrylamide (DMA) alkylation and bis(2-hydroxyethyl) disulphide (HED); an alternative procedure for the effective application of HE D is demonstrated. The benefits of precipitation and cup-loading versus in-gel rehydration were also assessed, with procedures for the employment of HED with the latter described. Following optimisation, a representative sample 21 proteins were identified from amniotic membrane using MS verify procedures were MS-compatible. Out results demonstrate that techniques for the reproducible separation of proteins from a proteinaceous structural tissue have been optimised. Briefly, proteins are extracted using a thiourea/urea extraction buffer containing carrier ampholytes, dithiothreitol (DTT), and 3-(cyclohexylamino)-1-propanesulfonic acid (CHAPS). After DMA alkylation, proteins were precipitated (using the 2-D clean-up kit from Amersham Biosciences) and resolubilised in extraction buffer containing a lower concentration of DTT. Samples were either cup-loaded onto rehydrated HED-containing strips or rebuffered into HED-containing buffer followed by in-gel rehydration.