Institute of Translational Medicine, Department of Pharmacy, Shenyang Medical College, Shenyang 110034, China.
ACS Appl Mater Interfaces. 2021 Mar 31;13(12):14028-14036. doi: 10.1021/acsami.1c02356. Epub 2021 Mar 17.
Aβ-conjugated magnetic nanoparticles, Aβ@MNP, were prepared by covalently coupling Aβ to hyperbranched polyethyleneimine (PEI)-modified magnetic nanoparticles via -(3-dimethylaminopropyl)-'-ethylcarbodiimide hydrochloride (EDC). Aβ's high binding capacity to glycosyl groups facilitates Aβ@MNP composite to be a promising selective adsorbent for glycoproteins in egg whites. In our study, under conditions of pH 4.0, the adsorption efficiency of Aβ@MNP composite for ovalbumin (100 μg mL) was 98.4% and its maximum adsorption capacity was 344.8 mg g ; under the condition of pH 4.0 and 200 mmol L NaCl, its adsorption efficiencies for ovalbumin and ovotransferrin were 96.9% and 60.0%, respectively. According to these primary data, in practice, ovalbumin was removed from egg white by Aβ@MNP composite at pH 4.0 (step I), and then after adding NaCl until the final salt concentration reached 200 mmol L (pretreated egg white), we utilized the same adsorbent to further isolate/purify glycoproteins (step II). SDS-PAGE results showed that Aβ@MNP composite could largely remove ovalbumin in step I and could isolate/purify the remaining ovalbumin and ovotransferrin in step II. LC-MS/MS analysis results showed that the removal of ovalbumin reduced its percentage in egg white samples from 32.93% to 11.05% in step I and the remaining ovalbumin and ovotransferrin were enriched in step II, where the final percentage reached 11.6% and 12.6%, respectively. In summary, 81 protein species were identified after two-step extraction with Aβ@MNP on egg white, while only 46 protein species were identified directly from raw egg white without any pretreatment. This work well illustrates the excellent adsorption performance of Aβ@MNP composite to glycoproteins and its potential in the application of proteomic studies on low-abundance proteins in egg white.
Aβ-缀合磁性纳米粒子（Aβ@MNP）通过盐酸 1-乙基-3-（3-二甲基氨基丙基）碳二亚胺（EDC）将 Aβ 共价偶联到超支化聚乙烯亚胺（PEI）修饰的磁性纳米粒子上制备。Aβ 与糖基的高结合能力使 Aβ@MNP 复合材料成为卵清中糖蛋白的一种很有前途的选择性吸附剂。在我们的研究中，在 pH 4.0 下，Aβ@MNP 复合材料对卵清白蛋白（100μgmL）的吸附效率为 98.4%，其最大吸附容量为 344.8mg g；在 pH 4.0 和 200mmolL NaCl 条件下，其对卵清白蛋白和卵转铁蛋白的吸附效率分别为 96.9%和 60.0%。根据这些初步数据，在实际应用中，通过 Aβ@MNP 复合材料在 pH 4.0 下（步骤 I）从卵清中去除卵清白蛋白，然后加入 NaCl 直至最终盐浓度达到 200mmolL（预处理卵清），我们利用相同的吸附剂进一步分离/纯化糖蛋白（步骤 II）。SDS-PAGE 结果表明，Aβ@MNP 复合材料在步骤 I 中可以很大程度上去除卵清白蛋白，并在步骤 II 中分离/纯化剩余的卵清白蛋白和卵转铁蛋白。LC-MS/MS 分析结果表明，在步骤 I 中，卵清白蛋白中卵清白蛋白的百分比从 32.93%降低到 11.05%，剩余的卵清白蛋白和卵转铁蛋白在步骤 II 中富集，最终百分比分别达到 11.6%和 12.6%。综上所述，通过 Aβ@MNP 在卵清上进行两步提取后，共鉴定出 81 种蛋白质，而直接从未经任何预处理的原始卵清中鉴定出的蛋白质只有 46 种。这项工作很好地说明了 Aβ@MNP 复合材料对糖蛋白的优异吸附性能及其在卵清白蛋白中低丰度蛋白质的蛋白质组学研究中的应用潜力。
