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.

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