Hopkinson Andrew, McIntosh Richard S, Layfield Robert, Keyte John, Dua Harminder S, Tighe Paddy J
University of Nottingham Division of Ophthalmology and Visual Sciences, EENT Centre, UK.
Proteomics. 2005 May;5(7):1967-79. doi: 10.1002/pmic.200401073.
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 prefractionation 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 HED 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. Our 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.
结构组织的蛋白质分析通常极具挑战性。羊膜具有独特的伤口愈合和抗瘢痕形成特性；然而，关于其活性蛋白成分却知之甚少。羊膜的结构性质使得整个二维（2-D）工作流程的开发和广泛优化成为必要。使用强大的增溶缓冲液提取蛋白质，并通过二维电泳进行分析，随后进行质谱（MS）鉴定。保存和处理导致可溶性蛋白与结构及膜相关蛋白的预分级分离。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）进行半胱氨酸封闭，实现了蛋白质溶解度的提高；文中展示了一种有效应用HED的替代方法。还评估了沉淀和杯上样与胶内复水的优缺点，并描述了将HED与后者结合使用的方法。优化后，使用MS验证程序从羊膜中鉴定出21种代表性蛋白质，这些程序与MS兼容。我们的结果表明，从蛋白质结构组织中可重复分离蛋白质的技术已得到优化。简而言之，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 - 丙烷磺酸（CHAPS）的硫脲/尿素提取缓冲液提取蛋白质。经过DMA烷基化后，蛋白质沉淀（使用Amersham Biosciences的2-D清理试剂盒），并在含有较低浓度DTT的提取缓冲液中重新溶解。样品要么杯上样到含有HED的复水条上，要么重新缓冲到含有HED的缓冲液中，然后进行胶内复水。
