Colby Thomas, Röhrig Horst, Harzen Anne, Schmidt Jürgen
Proteomics Unit, Max-Planck-Institut für Pflanzenzüchtungsforschung (MPIPZ), Köln (Vogelsang), Germany.
Methods Mol Biol. 2011;779:273-86. doi: 10.1007/978-1-61779-264-9_16.
Protein phosphorylation is a dynamic process of widespread regulatory significance. Phosphoproteomics attempts to provide a global view of this process during biological processes, but the approach is generally limited by the low relative amounts of phosphoproteins in biological samples. Although mass spectrometry (MS)-based technologies exist for the in-depth characterization of protein phosphorylation, these techniques are typically highly focused, have low throughput, and generally require special equipment and expertise. These specialized techniques are best used to support hypotheses generated by an initial broad-based survey, like the one described here. In this chapter, we outline a 2D gel-based phosphoproteomic methodology based on relatively inexpensive materials and basic, widely available MS technology. The goal is to provide a preparative and analytical laboratory framework that can generate the samples and hypotheses for phosphoproteomic MS, as well as a set of tools for biologically relevant phosphoproteomics for investigators who do not have ready access to phospo-MS technology. The combination of 2D gel-compatible metal-oxide affinity chromatography (MOAC)-based phosphoprotein enrichment and phospho-specific staining provides both the sensitivity necessary to make low-level phosphoproteins observable and identifiable, and the twofold phospho-selectivity to support their identity as phosphoproteins. An on-blot dephosphorylation assay for verifying the phospho-specificity of the enrichment method is also described here and provides a general tool for the validation of protein phosphorylation.
蛋白质磷酸化是一个具有广泛调节意义的动态过程。磷酸化蛋白质组学试图在生物过程中提供这一过程的全局视图，但该方法通常受到生物样品中磷酸化蛋白质相对含量较低的限制。尽管存在基于质谱（MS）的技术用于深入表征蛋白质磷酸化，但这些技术通常针对性很强、通量较低，并且一般需要特殊设备和专业知识。这些专门技术最适合用于支持由最初的广泛调查所产生的假设，就像这里所描述的那样。在本章中，我们概述了一种基于二维凝胶的磷酸化蛋白质组学方法，该方法基于相对廉价的材料和基本的、广泛可用的质谱技术。目标是提供一个制备和分析实验室框架，该框架可以为磷酸化蛋白质组学质谱分析生成样品和假设，同时为那些无法直接使用磷酸化质谱技术的研究人员提供一套用于生物学相关磷酸化蛋白质组学的工具。基于二维凝胶兼容的金属氧化物亲和色谱（MOAC）的磷酸化蛋白质富集与磷酸化特异性染色相结合，既提供了使低水平磷酸化蛋白质可观察和可识别所需的灵敏度，又提供了双重磷酸化选择性以支持它们作为磷酸化蛋白质的身份。本文还描述了一种用于验证富集方法磷酸化特异性的印迹上脱磷酸化测定法，并提供了一种用于验证蛋白质磷酸化的通用工具。
