This study used an Agilent HPLC-Chip coupled with an Agilent Accurate-Mass Q-TOF LC/MS to analyze the glycopeptide and glycan moiety of a monoclonal antibody (mAb). The tryptic peptide map of the mAb was generated and the glycopeptide was assigned using accurate mass measurement. The enzymatically released glycan moiety from the mAb was extracted and analyzed on an HPLC-Chip coupled with a Q-TOF. Diagnostic oxonium ions from a glycan moiety were further used to identify the glycopeptide and the glycan. The superior mass accuracy of the LC/MS platform, combined with the powerful data processing capabilities of Agilent MassHunter and BioConfirm software, enabled identification of the glycopeptides and glycan moiety attached to the protein complex.
This document details a study analyzing the glycopeptide and glycan moieties of a monoclonal antibody (mAb). The process involved generating a tryptic peptide map using an Agilent HPLC-Chip coupled with an Agilent Accurate-Mass Q-TOF LC/MS system. The glycan moiety was also enzymatically released and analyzed to confirm the glycopeptide identity using diagnostic oxonium ions.
The study utilized specific instrumentation, including an Agilent 1200 Series HPLC-Chip/MS Interface coupled with an Agilent 6520 Accurate-Mass Q-TOF LC/MS System. Data analysis was performed using Agilent MassHunter Qualitative Analysis, BioConfirm, and Spectrum Mill MS Proteomics Workbench software to identify glycopeptides and characterize the glycan structure.
| HPLC-Chip Enrichment Column | 40 nL |
|---|---|
| Analytical Column Dimensions | 75 mm x 43 mm |
| Flow Rate to Enrichment Column | 3 L/min |
| Flow Rate to Analytical Column | 600 nL/min |
| Sample Injection Amount | 50 ng |
| MS Resolution | 4 GHz |
| Veap Voltage | 1900 V |
| Fragmentor Voltage | 175 V |
This pretreatment ensures that the monoclonal antibody was completely denatured and soluble, allowing the protease to access its substrate efficiently.
The sample must be incubated at 37°C for 30 minutes.
Trypsin must be added to the solution at a ratio of 20:1 (protein to protease w/w).