Analytical & Testing Center of Beijing Normal University, Beijing 100875, China.
Analytical & Testing Center of Beijing Normal University, Beijing 100875, China.
Talanta. 2018 Aug 15;186:346-353. doi: 10.1016/j.talanta.2018.04.025. Epub 2018 Apr 22.
The tyrosine phosphorylation of proteins and peptides plays a vital role in cell signal transduction pathways, and it is very important to assay them for understanding their action mechanism. Due to the low levels of the tyrosine phosphopeptides (pTyr) in cells, it is a challenge to enrich them with traditional sorbents, therefore, development of specific and selective sorbents is urgent and necessary. In this work, the phosphate-imprinted magnetic nanoparticles (PMNPs) to enrich the pTyr with high efficiency and selectivity have been fabricated using the phenylphosphonic acid as a template for the "epitope" of pTyr. The magnetic nanoparticles have been functionalized with TiO and then the imprinting silica shells have been coated on the surface of the functional core to obtain the PMNPs sorbents. The PMNPs can obviously shorten the enrichment time and improve the adsorption efficiency for pTyr, and the epitope imprinting films provide an excellent selective recognition ability to target. The recognition capability of PMNPs for pTyr is 90.3 μg/mg and the imprinting factor of the sorbents can reach 24.4. The results indicate that the PMNPs can enrich the pTyr from the tryptic digest of β-casein samples with high specificity, and the spiking recoveries of the pTyr range from 85.1% to 93.8% with the RSD from 0.04 to 3.73. With the high adsorption capacity, rapid separation, excellent specificity and recyclability, the PMNPs sorbents show great potential for analysis of the phosphorylation of peptides in biological and medical fields.
蛋白质和肽的酪氨酸磷酸化在细胞信号转导途径中起着至关重要的作用，因此，研究它们的作用机制对于理解它们的作用机制非常重要。由于细胞中酪氨酸磷酸肽（pTyr）的含量较低，因此使用传统的吸附剂对其进行富集是一个挑战，因此，迫切需要开发特异性和选择性的吸附剂。在这项工作中，使用苯膦酸作为 pTyr 的“抗原决定簇”，制备了用于高效和选择性富集 pTyr 的磷酸化印迹磁性纳米粒子（PMNPs）。磁性纳米粒子用 TiO 功能化，然后在功能核表面涂覆印迹硅胶壳，得到 PMNPs 吸附剂。PMNPs 可以明显缩短富集时间，提高对 pTyr 的吸附效率，并且印迹的抗原决定簇印迹膜具有对靶标的优异选择性识别能力。PMNPs 对 pTyr 的识别能力为 90.3 μg/mg，吸附剂的印迹因子可达 24.4。结果表明，PMNPs 可以从 β-酪蛋白样品的胰蛋白酶消化物中特异性地富集 pTyr，pTyr 的加标回收率范围为 85.1%至 93.8%，相对标准偏差（RSD）为 0.04%至 3.73%。PMNPs 吸附剂具有高吸附容量、快速分离、优异的特异性和可重复使用性，在生物和医学领域的肽磷酸化分析中具有很大的应用潜力。
