We present a simple ratio method to infer protein composition within cellular structures using proximity labeling approaches but compensating for the diffusion of free radicals. We used tyramide signal amplification (TSA) and label-free mass spectrometry (MS) to compare proteins in nuclear speckles versus centromeres. Our “TSA-MS ratio” approach successfully identified known nuclear speckle proteins. For example, 96% and 67% of proteins in the top 30 and 100 sorted proteins, respectively, are known nuclear speckle proteins, including proteins that we validated here as enriched in nuclear speckles. We show that MFAP1, among the top 20 in our list, forms droplets under certain circumstances and that MFAP1 expression levels modulate the size, stability, and dynamics of nuclear speckles. Localization of MFAP1 and its binding partner, PRPF38A, in droplet-like nuclear bodies precedes formation of nuclear speckles during telophase. Our results update older proteomic studies of nuclear speckles and should provide a useful reference dataset to guide future experimental dissection of nuclear speckle structure and function.

中文翻译：

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酪酰胺信号放大质谱 (TSA-MS) 比率鉴定核斑点蛋白

我们提出了一种简单的比率方法，使用邻近标记方法来推断细胞结构内的蛋白质组成，但补偿自由基的扩散。我们使用酪酰胺信号放大 (TSA) 和无标记质谱 (MS) 来比较核斑点和着丝粒中的蛋白质。我们的“TSA-MS 比率”方法成功鉴定了已知的核斑点蛋白。例如，前 30 个和前 100 个分选蛋白质中的 96% 和 67% 的蛋白质分别是已知的核斑点蛋白，包括我们在此验证为富含核斑点的蛋白质。我们发现，在我们的列表中排名前 20 的 MFAP1 在某些情况下会形成液滴，并且 MFAP1 表达水平调节核斑点的大小、稳定性和动态。MFAP1 及其结合伴侣 PRPF38A 在末期核斑点形成之前定位在液滴状核体中。我们的结果更新了较早的核散斑蛋白质组学研究，并应提供有用的参考数据集来指导未来核散斑结构和功能的实验解剖。