Isolation And Detection Of Glycoproteins By Combining Monolithic Boronate Affinity Chromatography And Biological Mass Spectrometry

Protein glycosylation is a widely existing essential post-translational modification (PTM). Glycosylation play key roles in many biological processes. The occurrence of many diseases accompanies with the glycosylation state of related proteins. A variety of glycoproteins have been used as disease biomarkers for clinic diagnosis. Much effort has been paid on exploring methods for isolation and detection of glycoproteins. However, a convenient and fast method for the isolation and detection of glycoproteins is still missing. Also, little research has been focused on the isolation and detection of sialoglycoproteins, a sub-type of glycoproteins. Herein, we utilized boronate affinity materials to build a platform on which glycoproteins could be isolated and detected in a convenient and fast manner. In the meantime, the alternative detection of sialoglycoproteins or nonsialylated glycoproteins could be fulfilled.We chose a hydrophilic boronate affinity monolithic capillary as the isolation tool for glycoprotein. MALDI-TOF MS, which is a fast and direct detection method for bimolecular, was chosen as the detection tool. Since this is a hydrophilic boronate affinity monolithic capillary, we successfully utilized capillary action for sample loading which further simplifies the analysis process. So this monolithic capillary is not only an isolation tool, but also a portable loading tool. Convenient and fast detection of glycoproteins could be done on this platform.Herein, we present a new strategy for highly specifically extraction of sialoglycoproteins or nonsialylated glycoproteins. This strategy mainly relies on a finding that sialic acids exhibit anomalously high binding constants with common boronic acids when the pH is less than the pKa value of the boronic acid. First of all, we built a BAC-nano-ESI-Orbitap-MS platform for the alternative detection of sialic acid and neutral sugar. Then, a B AC-MALDI-TOF-MS platform was built for the alternative detection of sialylated and nonsialylated glycoproteins through pH manipulation. When the binding pH> the pKa of the boronic acid by one pH unit or more, the boronate affinity monolith selectively bind with glycoproteins containing neutral sugars and excludes sialic acid containing glycoproteins due to electrostatic repulsion. When the binding pH<the pKa by one pH unit or more, the boronate affinity monolith bind with sialylated glycoproteins due to the exceptional binding affinity of the boronic acid towards sialic acid residuals.Besides, we modified the target of MALDI-TOF MS with boronate affinity material and the on-line isolation and detection of glycoproteins could be fulfilled easily. We use two methods to modify the target. One is to modify the target with4-Mercaptophenylboronic acid utilizing the principle of self-assembly. The other is to apply molecular imprinting material on the target, and the isolation and detection of template glycoproteins could be accomplished on plate.