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.

Direct comparison of derivatization strategies for LC-MS/MS analysis ofN-glycans

“…The structural analyses were done using nano ESI with capillary voltage set to 1.6 kV, which was lower than the voltage added to ESI source (3.7 kV) that are commonly used for reducing end labeled glycans. 41, 42 Thus, the loss of sialic acid or the GlcNAc-Gal-Neu5Ac fragment is a commonly existed phenomenon in MS analysis of reducing end labeled glycans.…”

“…Tertiary amine groups are usually utilized in the tags, for example, procainamide (ProA), 14, 15 N 2 , N 2 , N 4 , N 4 -tetraethyl-6-hydrazinyl-1, 3, 5-triazine-2, 4-diamine (Meladrazine) 16 and RapiFluor-MS (RFMS) 17 all contain tertiary amines for increased ionization efficiency in positive ion mode. Quantitative glycomics can also be achieved using multiplexing reagent derivatization.…”

Simultaneous quantification of N- and O-glycans using a solid-phase method

“…This means that GIG can be scaled up for high-throughput analysis of glycans for quick screening of glycan biomarkers in clinical specimens. In addition, the N-glycans released by this high-throughput platform can be rapidly labeled with a fluorescent marker or an MS-active labeling reagent such as Rapifluor 52 for concurrent fluorescent detection (absolute quantification of glycans) and MS. As a variety of additional treatments can be incorporated into the procedure, GIG provides a platform for studying the structures of glycoproteins and glycans by corresponding glycosidases and glycosyltransferases.…”

Discovery and elucidation of unknown protein modifications : brominated coumarin azide as a probe for alkynes

“…Previously, a MS-compatible coumarin fluorophore tag with photoaffinity labeling was used to identify the ligand-binding site of a protein by leveraging fluorescence specificity over non-labeled peaks. 25 Fluorescent tags have also been used to label N-glycans to both identify and quantify the glycosylation profile in proteins 26 and employed to characterize the surface susceptibility of a monoclonal antibody. 27 However, distinguishing labeled from nonlabeled envelopes in MS is difficult in a complex matrix without a unique MS signature.…”

“…As shown in Figure 1-1, the alkyne and azide form a five-ring 1,2,3-triazole in the presence of a Cu(I) catalyst, generated by a Cu(II) salt and a reducing agent such as sodium ascorbate. [26][27][28] The concept of widely recognized "click chemistry" was first introduced by K.B. Sharpless in 2001 to describe reactions that give high yield, generate only by-products that can be removed without chromatographic methods, are wide in scope, stereospecific and simple to perform.…”