Glycosylation mainly refers to the enzymatic process that attaches glycans to proteins, lipids, or other organic molecules. It is the most common protein post-translational modifications (PTM). Glycosylation analysis is challenging because of the complexity and the isobaric nature of attached glycans. With the increasing application of protein therapeutics, such as monoclonal antibodies, glycoproteins, hormones, cytokines and clotting factors in clinical practice, routinely monitoring the N-glycosylation of proteins gains popular.
Figure 1. The different types of N-glycans produced in different organisms
N-glycans are heterogeneously antennated oligosaccharide structures with a Man3-GlcNAc2 core. They are linked to the polypeptide chain via a glycosidic bond between the terminal N-acetyl glucosamine (GlcNAc) and the NH2 group of Asparagine. The aim of this set of complementary analysis is to elucidate the global N-glycan profile of a molecule of interest (such as proteins or peptides). Most of N-glycan sample preparation consists of the general steps including digestion of the glycoprotein, de-glycosylation, purification, labelling/ derivatization and SPE. Additional steps and technical options could also be used depending on the nature of the samples.
Figure 2. The General workflow for N-glycans profiling
MALDI TOF MS and/or UHPLC based techniques determine the overall N-glycan population linked to a purified target molecule or a mixture of molecules.
1. N-Glycan profiling by MALDI-TOF MS
Qualitatively matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) represents high sensitivity and robustness for the structural characterization of glycosylated compounds and can also be used in a high-throughput manner. Single-charged ion formation maintained MALDI capability rather than electrospray ionization on profiling N-glycans mixtures. N-glycans released by enzymatic deglycosylation using Peptide N-glycosidase A or F is analyzed after permethylation.
Figure 3. Example of N-Glycan structures detected and analyzed by using MS
2. N-Glycan profiling by HILIC-UHPLC MS
Hydrophilic interaction chromatography (HILIC) has been a powerful technology for N-glycan profiling. So due to its wide acceptance, glycoprofiling of Enzymatic glycan release with PNGase F followed by derivatization by 2-aminobenzamide (2-AB) by HILIC-UHPLC is a fast and robust technique to analyze larger numbers of samples with well-known N-glycan profiles. Before fluorescent labelling, the harvested N-glycans should be further purified on a TSK Amide-80 column when large quantities of detergents, nonvolatile salts or materials with free amino groups are present which could interfere with the derivatization reaction. The labelled N-Glycans are then analyzed via HILIC separations combined mass spectrometric detection.
Figure 4. The workflow for N-glycan profiling using HILIC column
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