Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, and DAILAB , 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
ACS Appl Mater Interfaces. 2017 Jul 12;9(27):22970-22976. doi: 10.1021/acsami.7b05360. Epub 2017 Jun 28.
The identification of post-translational modifications (PTMs) in proteins has been of particular interest in the elucidation of human diseases and the improvement of therapeutic proteins. Herein, we report a novel strategy toward the construction of fingerprint-based PTM-sensing systems as an alternative to conventional specific recognition tools. Our strategy is based on poly-l-lysine (PLL) derivatives with two distinct properties suitable to fingerprint-based protein-sensing: (i) a turn-on fluorescent signal upon binding to proteins and (ii) condition-dependent cross-reactivity toward proteins and PTMs. One type of PLL derivative under varying solution properties (pH value and ionic strength) was sufficient to construct a sensing array that produces unique fluorescence fingerprints for structurally similar mammalian albumins with/without a wide variety of chemical modifications corresponding to PTMs. This approach was also applicable for the recognition of deviations in physicochemical properties of proteins as a result of realistic glycation and phosphorylation events. Multivariate analyses of the thus obtained fingerprints successfully identified analytes with 100% accuracy (qualitatively and quantitatively) in all cases. This study thus demonstrates for the first time a fingerprint-based sensing of proteins with/without PTMs using a single, highly accessible, and tunable synthetic polymer, and accordingly offers a powerful platform for simple high-throughput sensing of PTMs in proteins.
蛋白质翻译后修饰（PTMs）的鉴定一直是阐明人类疾病和改善治疗性蛋白质的研究热点。在此，我们报告了一种构建基于指纹的 PTM 传感系统的新策略，作为传统特异性识别工具的替代方法。我们的策略基于具有两种独特性质的聚赖氨酸（PLL）衍生物，适合基于指纹的蛋白质传感：（i）与蛋白质结合时开启荧光信号，（ii）对蛋白质和 PTM 具有条件依赖性的交叉反应性。在不同溶液性质（pH 值和离子强度）下的一种 PLL 衍生物足以构建一个传感阵列，该阵列为具有/不具有广泛化学修饰的结构相似的哺乳动物白蛋白产生独特的荧光指纹，这些修饰对应于 PTM。该方法还可用于识别由于实际糖基化和磷酸化事件导致的蛋白质物理化学性质的偏差。因此，通过对所得指纹进行多元分析，可以在所有情况下以 100%的准确率（定性和定量）成功识别分析物。这项研究首次使用单一、高度可及和可调的合成聚合物实现了基于指纹的 PTM 修饰蛋白的传感，因此为 PTM 在蛋白质中的简单高通量传感提供了一个强大的平台。
