Guoping Yao

Facile synthesis of aminophenylboronic acid-functionalized magnetic nanoparticles for selective separation of glycopeptides and glycoproteins

In this work, aminophenylboronic acid-functionalized magnetic nanoparticles were synthesized, and applied to selective separation of glycopeptides and glycoproteins.

On‐plate‐selective enrichment of glycopeptides using boronic acid‐modified gold nanoparticles for direct MALDI‐QIT‐TOF MS analysis

In this study, an on‐plate‐selective enrichment method is developed for fast and efficient glycopeptide investigation. Gold nanoparticles were first spotted and sintered on a stainless‐steel plate, then modified with 4‐mercaptophenylboronic acid to provide porous substrate with large specific surface and dual functions. These spots were used to selectively capture glycopeptides from peptide mixtures and the captured target peptides could be analyzed by MALDI‐MS simply by deposition of 2,5‐dihydroxybenzoic acid matrix. Horseradish peroxidase was employed as a standard glycoprotein to investigate the enrichment efficiency. In this way, the enrichment, washing and detection steps can all be fulfilled on a single MALDI target plate. The relatively small sample amount needed, low detection limit and rapid selective enrichment have made this on‐plate strategy promising for online enrichment of glycopeptides, which could be applied in high‐throughput proteome research.

Concanavalin A-immobilized magnetic nanoparticles for selective enrichment of glycoproteins and application to glycoproteomics in hepatocelluar carcinoma cell line.

Protein glycosylation is one of the most important PTMs in biological organism. Lectins such as concanavalin A (Con A) have been widely applied to N‐glycosylated protein investigation. In this study, we developed Con A‐immobilized magnetic nanoparticles for selective separation of glycoproteins. At first, a facile immobilization of Con A on aminophenylboronic acid‐functionalized magnetic nanoparticles was performed by forming boronic acid‐sugar‐Con A bond in sandwich structure using methyl α‐D‐mannopyranoside as an intermedium. The selective capture ability of Con A‐modified magnetic nanoparticles for glycoproteins was tested using standard glycoproteins and cell lysate of human hepatocelluar carcinoma cell line 7703. In total 184 glycosylated sites were detected within 172 different glycopeptides corresponding to 101 glycoproteins. Also, the regeneration of the protein‐immobilized nanoparticles can easily be performed taking advantage of the reversible binding mechanism between boronic acid and sugar chain. The experiment results demonstrated that Con A‐modified magnetic nanoparticles by the facile and low‐cost synthesis provided a convenient and efficient enrichment approach for glycoproteins, and are promising candidates for large‐scale glycoproteomic research in complicated biological samples.

Functionalized magnetic carbonaceous microspheres for trypsin immobilization and the application to fast proteolysis

In this study, magnetic carbonaceous (MC) microspheres prepared with a large-scale synthesis approach were developed as the novel substrate for enzyme immobilization, and the trypsin-immobilized MC microspheres were successfully applied to protein fast digestion. Firstly, MC microspheres with small size, strong magnetism, and biological compatibility were prepared through two-step solvothermal reactions. Secondly, MC microsphere surface was modified by 3-glycidoxypropyltrimethoxysilane (GLYMO). Finally, the enzyme was immobilized on the GLYMO-functionalized MC microspheres. The enzyme-immobilized magnetic microspheres were applied for fast protein digestion with microwave-assistance. Bovine serum albumin, myoglobin and cytochrome c, were used as model proteins to verify the digestion efficiency, and the digestion products were then characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with sequence coverage of 43%, 90% and 77%, respectively. The enzyme-immobilized magnetic particles were also successfully applied to the analysis of human pituitary extract. After database search, 485 proteins (p<0.01) were identified when the extract was digested by the microspheres. This opens a route for its future application in bottom-up proteomic analysis.

Facile synthesis of 4-mercaptophenylboronic acid functionalized gold nanoparticles for selective enrichment of glycopeptides.

In the work, 4-mercaptophenylboronic acid (4-MPBA)-functionalized gold nanoparticles were synthesized via a facile approach. At first, gold nanoparticles (about 50 nm) were prepared by a simple and convenient hydrothermal method based on a polyol process. Then, gold nanoparticles were modified with 4-MPBA by the well-known reaction of Au with the thiol groups. The MPBA-functionalized gold nanoparticles were characterized by Fourier transform infrared spectra and UV/Vis adsorption spectra. Due to the fact that the boronic acid group on the surface of 4-MPBA-modified gold particles can form tight yet reversible covalent bonds with glycopeptides containing cis-1,2-diols groups, the MPBA-modified gold nanoparticles were successfully applied to selective enrichment of glycopeptides. Isolation and enrichment of glycopeptides in a standard protein (asialofetuin and horseradish peroxidase) digestion and a complex sample were performed using MPBA-modified gold nanoparticles, followed by matrix-assisted laser desorption/ionization quadruple ion trap time-of-flight (MALDI-QIT-TOF) mass spectrometric analysis. The experimental results demonstrated that MPBA-modified gold nanoparticles synthesized by the facile approach have the powerful potential for selective enrichment of glycopeptides, and can be an alternative tool in glycoproteomics.