Agilent Technologies R&D and Marketing GmbH & Co KG, 76337Waldbronn, Germany.
Electrophoresis. 2010 Jan;31(4):611-7. doi: 10.1002/elps.200900346.
A novel pre-chip fluorescent derivatization method is presented for protein sizing and quantification by microchip CGE. The derivatization reaction employed a water-soluble and stable fluorescent dye and was performed under conditions that favored the formation of homogeneous reaction products. The method delivered in terms of protein sizing similar results as microchip CGE with on-chip staining but showed an extended linear dynamic range for protein quantification encompassing four orders of magnitude. The sensitivity of the method was similar to standard silver-stained planar gels. The characterization of derivatization reaction products by MS and preparative isoelectric focusing indicated that a constant degree of dye molecule tagging was obtained over a broad range of protein/dye ratios. The method allowed detecting and quantifying an impurity spiked into an antibody preparation down to a level of 0.05%. Advantages of this method compared with CGE approaches with pre-column derivatization include a shorter analysis time and an increased robustness and ease of use.
一种新颖的预芯片荧光衍生化方法被提出，用于通过微芯片 CGE 进行蛋白质大小测定和定量。衍生化反应采用了一种水溶性且稳定的荧光染料，并在有利于形成均相反应产物的条件下进行。该方法在蛋白质大小测定方面与微芯片 CGE 上的芯片内染色相比提供了相似的结果，但在蛋白质定量方面具有扩展的线性动态范围，涵盖了四个数量级。该方法的灵敏度与标准银染的平面凝胶相似。通过 MS 和制备等电聚焦对衍生化反应产物的表征表明，在广泛的蛋白质/染料比范围内获得了恒定的染料分子标记程度。该方法允许检测和定量掺入抗体制剂中的杂质，其浓度低至 0.05%。与预柱衍生化的 CGE 方法相比，该方法的优点包括分析时间更短，以及更高的稳健性和易用性。
