Haqqani Arsalan S, Kelly John F, Stanimirovic Danica B
Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada.
Methods Mol Biol. 2008;439:225-40. doi: 10.1007/978-1-59745-188-8_16.
A key issue in proteomics is to quantify changes in protein levels in complex biological samples under different conditions. Traditional two-dimensional gel (2-DE) electrophoresis-based proteomic approaches are tedious and suffer from several limitations, including difficulties in detecting low abundant and insoluble proteins. Isotope-coded affinity tagging (ICAT), one of the most employed chemical isotope labeling methods, can address many of the shortcomings of 2-DE. ICAT relies on the sensitivity of mass spectrometry (MS) to quantify relative protein abundance in a mixture of two differentially labeled protein samples. We describe here a detailed protocol for ICAT-based quantification of proteins in two or more biological samples, including sample preparation, ICAT labeling, fractionation and purification, and analysis by MS. For the MS analysis, we describe a "targeted" approach, which includes quantification of the samples using MS followed by selective identification of only the differentially expressed ICAT pairs using tandem MS (MS/MS). This approach gives more biologically relevant information than a data-dependent MS/MS analysis. We also describe the steps in data analysis, statistical analysis, and protein database searching.
蛋白质组学中的一个关键问题是量化不同条件下复杂生物样品中蛋白质水平的变化。传统的基于二维凝胶（2-DE）电泳的蛋白质组学方法繁琐且存在多种局限性，包括难以检测低丰度和不溶性蛋白质。同位素编码亲和标签（ICAT）是应用最广泛的化学同位素标记方法之一，它可以解决二维凝胶电泳的许多缺点。ICAT依赖于质谱（MS）的灵敏度来量化两个差异标记的蛋白质样品混合物中相对蛋白质丰度。我们在此描述一种基于ICAT对两个或更多生物样品中的蛋白质进行定量的详细方案，包括样品制备、ICAT标记、分级分离和纯化以及质谱分析。对于质谱分析，我们描述了一种“靶向”方法，该方法包括使用质谱对样品进行定量，然后仅使用串联质谱（MS/MS）选择性鉴定差异表达的ICAT对。与依赖数据的MS/MS分析相比，这种方法能提供更多与生物学相关的信息。我们还描述了数据分析、统计分析和蛋白质数据库搜索的步骤。
