Britton Chance Center for Biomedical Photonics at Wuhan National Laboratory for Optoelectronics - Hubei Bioinformatics & Molecular Imaging Key Laboratory, Systems Biology Theme, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China.
School of Chemistry, Chemical Engineering and Life Sciences, Wuhan University of Technology, Wuhan 430070, China.
J Chromatogr A. 2020 May 24;1619:460934. doi: 10.1016/j.chroma.2020.460934. Epub 2020 Jan 30.
Peptide-N-glycosidase F (PNGase F) is the most frequently used enzyme to release N-glycan from glycoproteins in glycomics; however, the releasing process using PNGase F is tedious and can range in duration from hours to overnight. Recently, efforts have been made to accelerate this enzymatic reaction, and they include the use of microwave irradiation, ultrahigh pressure, enzyme immobilization, and other techniques. Here, we developed a novel method combining the oriented immobilization of PNGase F on magnetic particles and microwave-assisted enzymatic digestion techniques to achieve highly efficient release of N-glycans. The oriented immobilization of PNGase F on magnetic particles utilizes the affinity of its co-expressed His-tag towards iminodiacetic acid-Nickel modified magnetic particles. Compared with non-oriented immobilization, the oriented immobilization of PNGase F exhibits several advantages including tolerance to high temperature (52 °C) and the ability to retain strong activity after more than five reuses. When used in combination with microwave irradiation, efficient N-glycan removal from ribonuclease B was achieved within 5 min. The proposed strategy was also used to release glycan from fetuin and human serum and has proven to provide a promising deglycosylation method for the characterization of protein glycosylation.
肽-N-糖苷酶 F（PNGase F）是糖组学中用于从糖蛋白中释放 N-聚糖最常用的酶；然而，使用 PNGase F 的释放过程繁琐，耗时从数小时到过夜不等。最近，人们努力加速这一酶促反应，包括使用微波辐射、超高压、酶固定化和其他技术。在这里，我们开发了一种将 PNGase F 定向固定在磁性颗粒上与微波辅助酶解技术相结合的新方法，以实现 N-聚糖的高效释放。PNGase F 在磁性颗粒上的定向固定利用了与其共表达的 His 标签对亚氨基二乙酸镍修饰的磁性颗粒的亲和力。与非定向固定相比，PNGase F 的定向固定具有耐高温（52°C）的优点，并且在重复使用五次以上后仍能保持较强的活性。当与微波辐射结合使用时，可在 5 分钟内从核糖核酸酶 B 中有效地去除 N-聚糖。所提出的策略还用于从胎球蛋白和人血清中释放聚糖，已被证明为蛋白质糖基化特征提供了一种有前途的去糖基化方法。
