Institute of Orthopaedic Biomedical and Device Innovation, School of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai, P. R. China.
Stomatological Hospital, School of Stomatology, Southern Medical University, Guangzhou, P. R. China.
J Sep Sci. 2024 Jul;47(13):e2400154. doi: 10.1002/jssc.202400154.
Glycosylation and phosphorylation rank as paramount post-translational modifications, and their analysis heavily relies on enrichment techniques. In this work, a facile approach was developed for the one-step simultaneous enrichment and stepwise elution of glycoproteins and phosphoproteins. The core of this approach was the application of the novel titanium (IV) ion immobilized poly(glycidyl methacrylate) microparticles functionalized with dendrimer polyethylenimine and phytic acid. The microparticles possessed dual enrichment capabilities due to their abundant titanium ions and hydroxyl groups on the surface. They demonstrate rapid adsorption equilibrium (within 30 min) and exceptional adsorption capacity for β-casein (1107.7 mg/g) and horseradish peroxidase (438.6 mg/g), surpassing that of bovine serum albumin (91.7 mg/g). Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was conducted to validate the enrichment capability. Experimental results across various biological samples, including standard protein mixtures, non-fat milk, and human serum, demonstrated the remarkable ability of these microparticles to enrich low-abundance glycoproteins and phosphoproteins from biological samples.
糖基化和磷酸化被认为是最重要的翻译后修饰，其分析严重依赖于富集技术。在这项工作中，开发了一种简便的一步同时富集和逐步洗脱糖蛋白和磷酸蛋白的方法。该方法的核心是应用新型钛（IV）离子固定化的树枝状多聚乙烯亚胺和植酸功能化的聚（甲基丙烯酸缩水甘油酯）微球。由于表面上丰富的钛离子和羟基，微球具有双重富集能力。它们表现出快速的吸附平衡（在 30 分钟内）和对β-乳球蛋白（1107.7mg/g）和辣根过氧化物酶（438.6mg/g）的卓越吸附能力，超过牛血清白蛋白（91.7mg/g）。此外，进行了十二烷基硫酸钠-聚丙烯酰胺凝胶电泳以验证富集能力。来自各种生物样品的实验结果，包括标准蛋白质混合物、无脂牛奶和人血清，证明了这些微球从生物样品中富集低丰度糖蛋白和磷酸蛋白的出色能力。
