Holland Ashling
Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford, OX1 3QX, Oxfordshire, UK.
Methods Mol Biol. 2018;1664:185-202. doi: 10.1007/978-1-4939-7268-5_15.
Comparative tissue proteomics aims to analyze alterations of the proteome in response to a stimulus. Two-dimensional difference gel electrophoresis (2D-DIGE) is a modified and advanced form of 2D gel electrophoresis. DIGE is a powerful biochemical method that compares two or three protein samples on the same analytical gel, and can be used to establish differentially expressed protein levels between healthy normal and diseased pathological tissue sample groups. Minimal DIGE labeling can be used via a 2-dye system with Cy3 and Cy5 or a 3-dye system with Cy2, Cy3, and Cy5 to fluorescently label samples with CyDye flours pre-electrophoresis. DIGE circumvents gel-to-gel variability by multiplexing samples to a single gel and through the use of a pooled internal standard for normalization. This form of quantitative high-resolution proteomics facilitates the comparative analysis and evaluation of tissue protein compositions. Comparing tissue groups under different conditions is crucially important for advancing the biomedical field by characterization of cellular processes, understanding pathophysiological development and tissue biomarker discovery. This chapter discusses 2D-DIGE as a comparative tissue proteomic technique and describes in detail the experimental steps required for comparative proteomic analysis employing both options of 2-dye and 3-dye DIGE minimal labeling.
比较组织蛋白质组学旨在分析蛋白质组在刺激下的变化。二维差异凝胶电泳（2D-DIGE）是二维凝胶电泳的一种改进和先进形式。DIGE是一种强大的生化方法，可在同一分析凝胶上比较两个或三个蛋白质样品，并可用于确定健康正常组织和患病病理组织样品组之间差异表达的蛋白质水平。通过使用含有Cy3和Cy5的双染料系统或含有Cy2、Cy3和Cy5的三染料系统，可在电泳前用CyDye荧光染料对样品进行最小化DIGE标记。DIGE通过将样品复用到单个凝胶上并使用混合内标进行归一化，避免了凝胶间的变异性。这种定量高分辨率蛋白质组学形式有助于对组织蛋白质组成进行比较分析和评估。通过表征细胞过程、理解病理生理发展和发现组织生物标志物，比较不同条件下的组织组对于推动生物医学领域的发展至关重要。本章讨论了作为比较组织蛋白质组学技术的2D-DIGE，并详细描述了采用双染料和三染料DIGE最小标记两种方法进行比较蛋白质组分析所需的实验步骤。
