Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Moscow, 119121, Russia.
Biochemistry (Mosc). 2012 Nov;77(11):1326-38. doi: 10.1134/S0006297912110120.
Use of small molecules for isolation of particular sub-proteomes is often complicated by the need for chemical modification of a parent compound for affinity sorbent preparation. Isatin (indoledione-2,3) is an endogenous indole that exhibits a wide spectrum of biological activities. Using 5-aminocaproylisatin for proteomic profiling of fractionated rodent brain homogenates, we previously identified more than sixty individual proteins. However, proteins tested in an optical biosensor study for validation of their isatin-binding capacity demonstrated different affinity for immobilized 5-aminocaproylisatin and 5-aminoisatin. In this study, we comparatively evaluated proteomic profiles of isatin-binding proteins separated using both isatin analogs as the affinity ligands. The total number of identified proteins was higher with the shorter isatin analog (88 versus 66), and only 22 proteins were identical in the two proteomic profiles. Thus, proteomic profiling of brain isatin-binding proteins is significantly influenced by the length of the spacer between the amino group used for affinity ligand coupling to Sepharose and the isatin moiety. This suggests that the actual number of brain proteins interacting with endogenous (unmodified) isatin still remains underestimated due to different affinity of proteins for the isatin analogs used for the affinity-based proteomic profiling.
使用小分子来分离特定的亚蛋白质组通常会受到需要对母体化合物进行化学修饰以制备亲和吸附剂的限制。色胺酮（吲哚二酮-2,3）是一种内源性吲哚，具有广泛的生物活性。我们曾使用 5-氨基己酰色胺酮对分馏的啮齿动物脑匀浆进行蛋白质组学分析，鉴定了六十多种不同的蛋白质。然而，在光学生物传感器研究中对用于验证其与色胺酮结合能力的蛋白质进行测试时，发现它们与固定化的 5-氨基己酰色胺酮和 5-氨基色胺酮的亲和力不同。在这项研究中，我们比较了使用这两种色胺酮类似物作为亲和配体分离的色胺酮结合蛋白的蛋白质组学图谱。使用较短的色胺酮类似物（88 个对 66 个）分离得到的鉴定蛋白质总数更高，且在两种蛋白质组学图谱中仅发现 22 个相同的蛋白质。因此，脑色胺酮结合蛋白的蛋白质组学分析受到用于将亲和配体偶联到琼脂糖上的氨基与色胺酮部分之间的间隔长度的显著影响。这表明，由于用于亲和蛋白质组学分析的色胺酮类似物的亲和力不同，与内源性（未经修饰）色胺相互作用的脑蛋白的实际数量仍然被低估。
