Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong.
Electrophoresis. 2013 Jul;34(13):1957-64. doi: 10.1002/elps.201200557.
Organelle-specific cell-permeable fluorescent dyes are invaluable tools in cell biology as they reveal intracellular dynamics in living cells. Mitrotracker is a family of dyes that strongly label the mitochondrion, a key organelle associated with many crucial cellular functions. Despite the popularity of these dyes, little is known about the molecular mechanism behind their staining specificity. Here, we aimed to identify the protein targets of one member of this dye family, mitotracker red (MTR), by 2DE and MS. MTR bound to cellular proteins covalently, and its fluorescence persisted even after cell lysis, protein solubilization, denaturation, and electrophoresis. This enabled us to display MTR-labeled proteins by 2DE. The MTR-specific fluorescent signals on the gel revealed the spots that contained MTR-conjugated proteins. These spots were analyzed by MS, resulting into the identification of ten proteins. We discovered that one major target is the mitochondrial protein HSP60 and that MTR staining could induce production of HSP60, predisposing cells to heat shock-like responses. The identification of the molecular targets of biological dyes, or "stainomics," can help correlate their intracellular staining properties with biochemical affinities. We believe this approach can be applied to a wide range of fluorescent probes.
细胞器特异性细胞通透荧光染料在细胞生物学中是非常有价值的工具，因为它们可以揭示活细胞内的细胞内动力学。Mitrotracker 是一类强烈标记线粒体的染料，线粒体是与许多关键细胞功能相关的关键细胞器。尽管这些染料很受欢迎，但对于它们染色特异性背后的分子机制知之甚少。在这里，我们旨在通过 2DE 和 MS 鉴定该染料家族的一个成员，即 mitotracker red（MTR）的蛋白靶标。MTR 与细胞蛋白共价结合，即使在细胞裂解、蛋白质溶解、变性和电泳后，其荧光仍然存在。这使我们能够通过 2DE 显示 MTR 标记的蛋白质。凝胶上的 MTR 特异性荧光信号揭示了含有 MTR 缀合蛋白的斑点。通过 MS 对这些斑点进行分析，鉴定出了十种蛋白质。我们发现一个主要靶标是线粒体蛋白 HSP60，并且 MTR 染色可以诱导 HSP60 的产生，使细胞易发生类似热休克的反应。生物染料的分子靶标的鉴定，或“染色组学”，可以帮助将其细胞内染色特性与生化亲和力相关联。我们相信这种方法可以应用于广泛的荧光探针。
