N-Glycan Profiling Service | HILIC-UHPLC MS

Glycosylation primarily refers to the enzymatic process of linking polysaccharides to proteins, lipids, or other organic molecules, and is the most common type of post-translational modifications (PTMs). Due to the complexity and isoform nature of the linked polysaccharides, glycan analysis presents significant challenges. As protein therapeutics such as monoclonal antibodies, glycoproteins, hormones, cytokines, and coagulation factors are increasingly used in clinical practice, the detection of protein N-glycosylation (also known as N-glycan, N-sugar) has received growing attention.

 

N-glycan is an oligosaccharide structure containing Man3-GlcNAc2, which is linked to the polypeptide chain of proteins through a glycosidic bond between the terminal N-acetylglucosamine (GlcNAc) and the NH2 group of asparagine. The sample preparation steps for most glycan analysis include glycoprotein digestion, deglycosylation, purification, labeling/derivatization, and SPE. Depending on the characteristics of the sample, other steps and techniques may also be used for sample preparation.

 

Utilization of MALDI-TOF-MS and/or UHPLC Techniques for Glycan and Type Analysis

1. Based on MALDI-TOF-MS Technique for N-glycan Analysis

MALDI-TOF offers high sensitivity in structural characterization of glycosylated compounds and allows for high-throughput detection. In the analysis of mixture N-glycans, MALDI performs the analysis by forming single charged ions, rather than using electrospray ionization. After releasing the N-glycans from the sample using N-glycosidase A or F, the sample is methylated for detection.

 

2. Based on HILIC-UHPLC-MS Technique for N-glycan Profiling Analysis

LC-MS-based N-glycan analysis is the current mainstream analytical method. Hydrophilic interaction chromatography (HILIC) is suitable for N-glycan analysis. N-glycans released by the PNGase F enzymatic reaction are derivatized using 2-aminobenzamide (2-AB), and the derivatized glycans are measured for large sample N-glycan analysis. Prior to fluorescence labeling, if there are detergents, non-volatile salts, or substances with free amino groups that might interfere with the derivatization reaction, the N-glycans can be purified using a TSK Amide-80 chromatography column. The labeled N-glycans are then analyzed by HILIC-UHPLC-MS.

 

MtoZ Biolabs has much analytical service experience in glycomics and can customize glycomics analysis services according to your specific needs. Free consultation is available!

 

Deliverables

1. Experimental Procedures

2. Mass Spectrometric Parameters

3. Mass Spectrometric Images

4. Raw Data

5. N-glycan Analysis Results