Moreda-Piñeiro Antonio, García-Otero Natalia, Bermejo-Barrera Pilar
Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Chemistry, University of Santiago de Compostela, Avenida das Ciencias, s/n. 15782 Santiago de Compostela, Spain.
Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Chemistry, University of Santiago de Compostela, Avenida das Ciencias, s/n. 15782 Santiago de Compostela, Spain.
Anal Chim Acta. 2014 Jul 11;836:1-17. doi: 10.1016/j.aca.2014.04.053. Epub 2014 May 1.
Mass spectrometry (MS) techniques are commonly used for protein identification and further analysis of selected protein spots after high resolution 2-D electrophoresis. Complementary gel-free approaches have been developed during the last few years and have shown to be useful tools in modern proteomics. The development and application of various gel-free electrophoresis devices for performing protein fractionation according to the pI differences is therefore a topic of interest. This review describes the current state of isoelectric focusing (IEF) gel-free electrophoresis based on the Agilent offgel 3100 fractionator. The review includes, therefore, (i) an overview on IEF as well as other previous IEF gel-free electrophoresis developments; (ii) offgel fundamentals and future trends; (iii) advantages and disadvantages of current offgel procedures; (iv) requirements of isolated protein pellets for further offgel fractionation; (v) offgel fraction requirements to perform the second dimensional analysis by advance electrophoresis and chromatographic techniques; and (vi) effect of the offgel operating conditions on the stability of metal-protein complexes.
质谱（MS）技术通常用于蛋白质鉴定以及在高分辨率二维电泳后对选定蛋白质斑点进行进一步分析。在过去几年中，已开发出互补的无凝胶方法，并且已证明这些方法是现代蛋白质组学中的有用工具。因此，开发和应用各种基于pI差异进行蛋白质分离的无凝胶电泳装置是一个备受关注的话题。本综述描述了基于安捷伦Offgel 3100分级分离器的等电聚焦（IEF）无凝胶电泳的现状。因此，本综述包括：（i）IEF以及其他先前IEF无凝胶电泳发展的概述；（ii）Offgel基本原理和未来趋势；（iii）当前Offgel程序的优缺点；（iv）用于进一步Offgel分级分离的分离蛋白质沉淀的要求；（v）通过先进的电泳和色谱技术进行二维分析所需的Offgel分级分离要求；以及（vi）Offgel操作条件对金属 - 蛋白质复合物稳定性的影响。
