Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
Key Laboratory of Proteomics, Dalian Medical University, Dalian, China.
Methods Mol Biol. 2021;2237:207-215. doi: 10.1007/978-1-0716-1064-0_17.
Reverse phase protein array (RPPA), a high-throughput, parallel immunoassay in a dot-blot format, is a powerful tool to quantitatively profile protein expression in multiple samples simultaneously using small amounts of material. Despite its success, analysis of post-translationally modified (PTM) proteins has been limited in RPPA assays, primarily due to relatively low availability of antibodies specific to proteins of PTMs, e.g., glycosylation. Moreover, the high matrix complexity, with tens of thousands of proteins in cell lysates or tissue extracts and the low abundance of proteins with PTMs, makes it extremely challenging to detect these proteins with PTMs. Therefore, there is an urgent need to fill this gap, which would greatly contribute to the analysis of a specific PTM by RPPA. In this chapter, we introduce a novel RPPA platform, termed polymer-based reverse phase glycoprotein array (polyGPA), to measure the variation of glycosylation patterns on a three-dimensionally functionalized RPPA. Without the need of specific antibody towards glycosylation, polyGPA represents a highly sensitive strategy to analyze protein glycosylation in multiple complex biological samples in parallel.
反转蛋白阵列（RPPA）是一种高通量、平行免疫检测点印迹格式，是一种强大的工具，可同时使用少量材料定量分析多个样本中的蛋白质表达。尽管取得了成功，但 RPPA 检测中对翻译后修饰（PTM）蛋白的分析一直受到限制，主要是因为针对 PTM 蛋白（例如糖基化）的抗体相对较少。此外，细胞裂解物或组织提取物中存在成千上万种蛋白质，基质复杂性高，而具有 PTM 的蛋白质丰度低，这使得检测这些具有 PTM 的蛋白质极具挑战性。因此，迫切需要填补这一空白，这将极大地有助于通过 RPPA 分析特定的 PTM。在本章中，我们介绍了一种新型的 RPPA 平台，称为基于聚合物的反转蛋白糖基化阵列（polyGPA），用于测量三维功能化 RPPA 上糖基化模式的变化。由于不需要针对糖基化的特异性抗体，polyGPA 代表了一种在多个复杂生物样本中平行分析蛋白质糖基化的高度敏感策略。
