Among PTM analyses, characterization of glycosylation is of prime importance for the development of a recombinant drug. Quality, quantity and reproducibility of glycosylation have to be demonstrated from R&D to production of the molecule.

In addition, establishing glycosylation profiles of complex samples (plasma, tissue extracts) in a glycomics approach, may allow discovering new biomarkers for diagnosis and treatment of diseases. This is why we have developed a set of complementary techniques that allow determining glycosylation. These analyses are based on mass spectrometry (MS) techniques that are approved by regulatory agencies.

N-glycosylation Analyses

N-glycans are heterogeneous antennated oligosaccharide structures with a Man₃-GlcNAc₂ core. They are linked to the polypeptide chain via a covalent bond between the terminal GlcNAc and the NH₂ group of asparagin.

Determination of the N-glycan
profile of purified molecules

The aim of this study is to elucidate the global N-glycan profile of a molecule of interest (protein, peptide). This technique allows to determine the overall N-glycan population linked to a target molecule and to compare its profile to a model (native protein or theoretical values). It is also used to characterize the glycosylation of a production lot and to qualify reproducibility of a production process. We have developed efficient tools for sample preparation (native or permethylated) that are compatible with glycan analysis by MS. Chemical modification of N-glycans by permethylation allows semi-quantitative analysis based on MS (relative abundance of N-glycans).

Determination of N-glycan profiles from complex samples

The comparison of glycan profiles from complex samples (plasma from patients compared to plasma from healthy donors, protein extracts from tumor tissue compared to healthy tissue) in a “glycomics” approach, allows to discover new biomarkers or to follow effects of a specific treatment.

N-glycan sequencing
by MS/MS fragmentation

Confirmation of the structure of a specific glycan by ESI-MS/MS, MSⁿ or MALDI-PSD fragmentation Fragmentation allows to confirm the linkage between different sugars or the branching of antennae and to determine the presence of specific structures (Gal-α 1 ,3-Gal epitope or sialic acid NAGA).

 

Analysis of N-glycosylation site occupation

Occupation of N-glycosylation sites is determined by combining different peptide maps of a target protein after:

• Complete protease digest of the protein,
• Enrichment of the N-glycopeptide fraction on a lectin column
  with and without PNGase digest,
• Non glycosylated peptide fraction after lectin column.

Monosaccharide composition

Monosaccharide composition is determined by GC-MS after total chemical hydrolysis and chemical modification of the sugar structures. This technique allows determining relative quantities of each monosaccharide present on the analyzed molecule. It is also used to identify the presence of specific sugars such as xylose and GalNAc.

Lectin based analyses

Specific lectins are used for example to determine the type of linkage of sialic acids, to confirm the presence of the Gal-α-Gal epitope or for other types of studies. These analyses are performed in collaboration with our partner GLYCODIAG.

O-glycosylation Analyses

O-glycans are divided into several structural families and are relatively heterogeneous. Biosynthesis of O-glycans is less well elucidated than biosynthesis of N-glycans. O-glycans are linked to the protein mainly through a covalent bond between the terminal GalNAc residue and the OH group of serine or threonine. We use different techniques for free O-glycan or O-glycopeptide analysis to determine the O-glycosylation profile of a target molecule.