The presence of sialic acids is one characteristic of glycosylated therapeutic proteins. The presence of these charged monosaccharides is critical for the immunogenicity properties and structural properties of the proteins. Profiling of the N-glycans and their charge state is a requisite for complete protein characterization. Two analytical methods developed on released N-glycans are described in this chapter, allowing for the determination of the sialoglycosylation with different levels of details. In the first method (AEX-HILIC/FLR), N-glycans are separated based on their charge and the average charge state can be determined from the fluorescence profile. In the second method (AEX-RP-FLR-MS), N-glycans are also separated based on their charge and the sialylation level is determined based on the fluorescence signal. In addition, in this method, the N-glycans are also separated by type and identified with the hyphenated MS. For both methods, an optimized protocol with fast and high-throughput sample preparation and purification is presented.
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Article Synopsis
- Comprehensive enrichment of N-glycans and associated molecules is crucial in N-glycomics research to simplify samples, reduce impurities, and enhance analysis quality.
- The research focuses on a specific Fbs1 GYR variant (Fg), which has a strong binding affinity for the core pentasaccharide of N-glycans, making it effective for this purpose.
- The study involved cloning the Fg into a GST-tagged vector for easy purification, resulting in a stable and effective protein that can efficiently isolate N-glycans and their derivatives for analytical applications.
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