Case Study of Biopharmaceutical Glycosylation Detection

Glycosylation is an important post-translational modification of proteins. Glycosylation of

proteins can be classified into N-glycosylation and O-glycosylation depending on the manner in
which the sugar chain and the peptide chain are linked.

N-glycosylation is linked by the N-acetylglucosamine (Glc-NAc) at the reducing end of the sugar
chain to the nitrogen atom on the side chain acylamino group of some Asn in the peptide chain.
The Asn which can be attached to the sugar chain must be in the motif consisting of the
Asn-X-Ser/Thr 3 residue, where X can be any amino acid residue other than Pro. The structure of
O-glycosylation is simpler than N-glycosylation, and the sugar chain is generally shorter, but the
species is much more numerous than N-glycosylation. The main glycosylation in the peptide chain
is Ser and Thr, in addition to tyrosine, hydroxylysine and hydroxyproline, and the linked sites are
the hydroxyl oxygen atoms on the side chains of these residues. Based on the high-resolution mass
spectrometer Obitrap Fusion Lumos, MtoZ Biolabs, an international contract research
organization (CRO) providing advanced proteomics, metabolomics, bioinformatics related
services, uses glycin software containing almost all glycoforms for glycosylation of peptides,
proteins and antibody drugs to identify peptide or protein sample glycosylation sites. , N sugar / O
sugar type, glycosylation site sugar composition.

Case Study
The service case is a glycan assay for a recombinant protein drug provided by a client. After
obtaining the theoretical sequence provided by the client, MtoZ Biolabs first analyzed the
sequence to confirm the protease suitable for the project. Usually, we would choose two or more
proteases for enzyme digestion to improve the peptide coverage. The protease is digested to
produce different peptide sequences, and the complementarity of the peptide sequences is used to
help improve the peptide coverage of the glycan analysis. The following is part of the
experimental data from the previous project. The customer provided a sample of recombinant
protein. After the appropriate protease combination is selected , the peptide coverage is shown in
the following figure:

In this project, the peptide coverage was 94% compared to the theoretical sequence, and the
unidentified 6% sequence was a signal peptide, which was cut off during the secretion of the
recombinant protein, so the actual peptide coverage was 100%.

After the analysis by Byonic software, the identified glycopeptides are summarized as follows:
In the final report, MtoZ Biolabs also provides secondary mass spectrometry information for each
glycopeptide identified as shown in the following figure:

In the report, MtoZ Biolabs provided customers with detailed technical reports in both English and
other languages needed, including experimental steps, related mass spectrometry parameters, mass
spectrum image, raw data, glycan detection results.