Sugar Chain Structural Analysis
Sugar Chain Structural Analysis of Mouse Monoclonal Antibodies
As the sugar chains in glycoproteins such as antibody drugs are changed by the actions of multiple enzymes after protein translation, the diversity and nonuniformity of the sugar chain structure is an unavoidable problem. Nevertheless, the guidelines require the analysis of the sugar chain structures to the maximum extent possible. As the relationship between the existence of fucose (one component of sugar chains) and antibody-dependent cellular cytotoxicity (ADCC) has been reported, for example, the importance of analyzing sugar chains in antibody drugs for research and development will increase in the future.
This is an example of the sugar chain structural analysis of commercial mouse monoclonal antibodies.
Test and research flow
1. Use SDS-PAGE to excise bands corresponding to antibodies from the stained gel.
2. Perform in-gel digestion using PNGaseF.
3. Purify sugar chains and apply pyridylamino (PA) markers using the BlotGlyco kit (Sumitomo Bakelite).
4. Perform MSn analysis using Prominence nano/AccuSpot/AXIMA Resonance.
Table 1 shows the detected ion m/z values and the elution times from Prominence nano. The m/z values are expressed as mean mass values (integer values). Seven ions were detected: m/z 1418, m/z 1580, m/z 1564, m/z 1726, m/z 1888, m/z 2047, and m/z 2209.
Fig. 1 shows the MS/MS and MS/MS/MS spectra of m/z 1580 and m/z 1726. The predicted sugar chain structures are shown in the diagram. to the software can easily determine the existence of fucose, which is implicated in ADCC.
In addition to the sugar chains in Fig. 1, the existence of other sugar chains was confirmed with structures such as G0-F, G0, G1'-G1', G2, G0+S, and G2+S.
As this analysis used antibodies excised from the gel as the starter substance, Prominence nano/AccuSpot was used for trace analysis.
- Prominence nano LC permits highly sensitive sugar chain analysis. The unique reflux flow control system enhances separation reproducibility, and the dedicated Nano-Assist software simplifies automation and parameter settings.
- AccuSpot automatically adds the matrix and spots the sugar chains separated by the Prominence nano system onto MALDI plates.
- Separation and purification of sugar chain mixtures by Prominence nano reduces ion suppression by impurities.
- Analysis software provides powerful support for sugar chain structural analysis.