Therapeutic Antibody Glycopeptides Analysis Services

Antibodies used in biopharmaceuticals are usually modified with glycans. The glycan is a molecule with high structural heterogeneity, composed of complex coupled monosaccharides (such as glucose and mannose), and it is known that the complex structure plays an important role in the function regulation of proteins. Since mAbs are heterogeneous molecules in nature, it is very important to clarify the distribution and composition of various glycans when antibodies are biosynthesized as biopharmaceuticals. Mass spectrometers are now commonly used to determine binding sites and glycan structures. At Creative Biolabs, we use high-resolution technology with unique high-energy fragmentation capabilities to determine the structure and binding sites of glycopeptides bound to antibodies.

Preparation of glycopeptides from monoclonal antibodies

First, use a commercially available monoclonal antibody as a sample, and perform reduction and alkylation in a solution. Then, trypsin was added to the solution, and the enzymatic digestion was continued for 2 hours. Then the enzyme digested solution was added to the spin column. The gel was equilibrated with butanol: ethanol: water (4:1:1) in advance, and then the solution was allowed to interact with the gel. The glycopeptide is adsorbed to the gel. Then, the non-glycosylated peptide is removed by washing with an equilibrium solution, and the glycopeptide is recovered using an aqueous ethanol solution. Using 2,5-dihydroxybenzoic acid (DHB) as a matrix, the recovered glycopeptides were spotted on the target plate and analyzed by MS.

Mass spectrometry analysis of glycopeptides

The mass spectrum of the recovered glycopeptide portion is shown in Figure 1. Among the detected signals, m/z 2405.72, 2430.75, 2567.72, 2592.73, 2635.74, 2795.74 and 2957.74 were considered as glycopeptide-derived signals. Because glycans have a non-uniform structure, there are multiple different glycan structures in a peptide backbone. Since the signals are related to monovalent ions, the mass differences between the peaks can be viewed. If they match the mass of the glycan moiety, these peaks are most likely glycopeptides.

Fig.1 Intensity of glycan oxonium ions differs between higher-energy collisional dissociation (HCD) fragmented O- and N-glycopeptides.^{1, 2}

With extensive experience in glycan characterization, our team can work on custom solutions to meet the unique requirements of each product. With flexible global capabilities, you can provide services where you need it most. Contact our expert scientists today to discuss how our range of glycan analytical services can improve your unique process.

References

1. Toghi Eshghi, Shadi, et al. "Classification of tandem mass spectra for identification of N-and O-linked glycopeptides." Scientific Reports 6.1 (2016): 1-8.
2. under Open Access License CC BY 4.0, without modification.