Abstract:

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.

Key words: boronate affinity, glycoproteins, molecular recognition, nanoparticles, size exclusion