Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Labeling Reagent that Facilitates Sensitive Fluorescence and ESI-MS Detection.

Abstract

N-glycosylation of proteins is now routinely characterized and monitored because of its significance to the detection of disease states and the manufacturing of biopharmaceuticals. At the same time, hydrophilic interaction chromatography (HILIC) has emerged as a powerful technology for N-glycan profiling. Sample preparation techniques for N-glycan HILIC analyses have however tended to be laborious or require compromises in sensitivity. To address these shortcomings, we have developed an N-glycan labeling reagent that provides enhanced fluorescence response and MS sensitivity for glycan detection and have also simplified the process of preparing a sample for analysis. The developed labeling reagent rapidly reacts with glycosylamines upon their release from glycoproteins. Within a 5 min reaction, enzymatically released N-glycans are labeled with this reagent comprised of an NHS-carbamate reactive group, a quinoline fluorophore, and a tertiary amine for enhancing ESI+ MS ionization. To further expedite the released N-glycan sample preparation, rapid tagging has been integrated with a fast PNGase F deglycosylation procedure that achieves complete deglycosylation of a diverse set of glycoproteins in approximately 10 min. Moreover, a technique for HILIC-SPE of the labeled glycans has been developed to provide quantitative recovery and facilitate immediate HILIC analysis of the prepared samples. The described approach makes it possible to quickly prepare N-glycan samples and to incorporate the use of a fluorescence and MS sensitivity enhancing labeling reagent. In demonstration of these new capabilities, we have combined the developed sample preparation techniques with UHPLC HILIC chromatography and high sensitivity mass spectrometry to thoroughly detail the N-glycan profile of a monoclonal antibody.