Low-molecular-weight glycoproteins (LMW-GPs) are considered promising candidates for disease biomarker discovery. Selective sorbents are essential for the extraction and enrichment of this class of compounds. Boronate affinity chromatography is a unique separation mode in liquid chromatography. It enables the selective separation and isolation of cis-diol-containing compounds such as glycoproteins and saccharides. Recent years have witnessed the rapid development of boronate affinity materials, particularly for use as selective sorbents in proteomics and metabolomics. However, studies are scarce on the specific design of such materials for the selective extraction of LMW-GPs. Herein, we present multifunctional magnetic nanoparticles (MNPs) for selectively harvesting LWM-GPs. The multifunctional MNPs were rationally designed and prepared by wrapping magnetic core nanoparticles with a phenylboronic acid-grafted poly(acrylic acid) (PAA) network. In addition to fulfilling the primary function of conventional MNPs in magnetic separation, multifunctional MNPs can offer three pre-determined advanced functions: 1) the size-restriction effect, which enables the elimination of the interference of high-molecular-weight proteins and other species; 2) the selective extraction of LMW-GPs; and 3) protection of the harvested LMW-GPs against degradation and contamination. The multifunctional MNPs enable selective extraction due to the affinity of the boronic acid ligand to the cis-diol moieties of the glycoproteins. The size-restriction effect and protection function depend on the polymer network on the surface of the MNPs, which allows the selective passage of low-molecular-weight molecules. Transmission electron microscopy (TEM) characterization showed that the MNPs were well-shaped nanoparticles, with a diameter of approximately 60 nm. The size-restriction effect was first predicted by a thermogravimetric analysis-based theoretical calculation, where for MNPs prepared using PAA with an average molecular weight of 240 kDa, the estimated pore size of the network was 0.9 nm. The boronate affinity and size-exclusion effect were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and capillary zone electrophoresis (CZE). To investigate the dependence of the selectivity of the MNPs to LMW-GPs in a complex environment and the size-restriction threshold for the PAA chain length, nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) was performed to analyze the molecular mass of fragments harvested by the MNPs from the tryptic digest of horseradish peroxidase (HRP, a typical glycoprotein). The polymer chain length or the molecular weight of the PAA used played a critical role in determining the molecular weight thresholds of proteins above which the size exclusion effect will occur. The threshold values were found to be 5.0, 9.3, 4.1, 5.1, and 2.7 kDa for MNPs prepared using PAA with average molecular weights of 2, 5, 15, 100, and 240 kDa, respectively. This dependence enabled adjustment of the threshold value for inducing the size-exclusion effect of the multifunctional MNPs by changing the PAA chain length. The multifunctional MNPs can be further developed into promising nanoprobes for selectively harvesting not only LMW-GPs, but also other cis-diol-containing biomolecules of biological importance, such as nucleosides and glycans. Thus, the material preparation strategy reported herein offers new insights for the rational design and synthesis of multifunctional-affinity sorbents to selectively extract target compounds from a complex sample matrix.
低分子量糖蛋白被认为是发现疾病生物标志物的宝库。特异性的萃取吸附剂对这一类化合物的萃取和富集是必不可少的。硼亲和材料在近年来取得了很大的发展,但专门用于选择性富集低分子量糖蛋白的硼亲和材料目前鲜有报道。该文提出了具有多种功能的磁性纳米颗粒(MNPs),用于低分子量糖蛋白的选择性捕获。该多功能磁性纳米颗粒是用硼酸功能化聚合物网络包裹的磁性纳米复合物。该多功能磁性纳米材料是利用磁性的纳米颗粒内核通过在其表面修饰苯硼酸功能团的聚丙烯酸高分子网络链制备得到。该材料不仅具有常规磁性材料在磁分离方面的基本优势,还能提供三重预先设计的先进功能:1)尺寸排阻效应,去除高分子量蛋白质的干扰;2)对低分子量糖蛋白的选择性萃取;3)保护捕获到的低分子量糖蛋白不被降解和污染。该材料的选择性萃取功能来自于硼酸配基与糖蛋白的顺式二醇部分的亲和性,而尺寸限制效应和保护功能则依赖于磁性纳米颗粒表面修饰的聚合物网络,允许低分子量化合物选择性通过。通过实验验证了这些预设的功能,且通过改变聚合物链长可以调节限径效应的阈值。这种多功能磁性纳米复合物可以进一步发展成有前景的纳米探针,不仅可以选择性捕获低分子量糖蛋白,还可以选择性捕获核苷和聚糖等其他具有重要生物学意义的顺式二醇分子。因此,该文报道的材料制备策略为从复杂样品中选择性萃取靶标化合物的多功能吸附剂的设计和合成提供了新思路。
Keywords: boronate affinity; glycoproteins; molecular recognition; nanoparticles; size exclusion.