Sugar Chain Structural Analysis (LCMS-IT-TOF)

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Analysis of Commercial Lab Mouse Monoclonal Antibodies

As the sugar chains in glycoproteins such as antibody drugs are added by the actions of multiple enzymes after protein translation, the diversity and nonuniformity of the sugar chain structure is an unavoidable problem. The Guidelines require the analysis of the sugar chain structures to the maximum possible extent. 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 sugar chains in antibody drugs for research and development will increase in the future.

Experiment and research flow

  1. Conduct SDS-PAGE and excise the bands equivalent to antibodies from the stained gel.
  2. Conduct in-gel digestion using PNGaseF.
  3. Purify sugar chains using BlotGlyco kit (manufactured by Sumitomo Bakelite Co.,Ltd.) and add pyridylamino (PA) markers.
  4. Perform MSn analysis using Prominence nano/LCMS-IT-TOF.

Results

Fig. 1 MS Chromatogram by nanoLCMS-IT-TOF

Fig. 1 shows the mass chromatograms of m/z 696.7672, m/z 769.2833, m/z 850.3369, and m/z 931.3477.

Fig. 2 and Fig. 3 show the MS/MS spectra using precursor ions from the mass chromatogram in Fig. 1.
The sugar chain structures predicted by the sugar chain structure prediction software are shown in the diagram.

The system is able to easily determine the existence of fucose, which is implicated in antibody-dependent cellular cytotoxicity (ADCC).

In addition to the sugar chains in Fig. 2 and Fig. 3, the existence of other sugar chains with structures G1 and G2 was confirmed.

As this analysis used antibodies excised from the gel as the starter substance, Prominence nano/LCMS-IT-TOF was used for trace analysis.

Fig. 2 MS/MS Spectrum and Predicted Sugar Chain Structures (Precursor ion m/z 696.7672[M-2H]2-)

Fig. 3 MS/MS Spectrum and Predicted Sugar Chain Structures (Precursor ion m/z 769.2833[M-2H]2-)

High-Performance Liquid Chromatograph Mass Spectrometer

sample
  • The LCMS-IT-TOF High-Performance Liquid Chromatograph-Mass Spectrometer produces MSn information with high mass accuracy that is ideal for sugar chain analysis.
  • A combination of the Prominence nano Nanoflow High-Performance Liquid Chromatograph with the NES-100 NanoESI Interface offers highly sensitive sugar chain analysis.
  • The high mass accuracy of the LCMS-IT-TOF allows highly accurate sugar chain estimation.
  • The neutral loss survey automatically evaluates preset specific neutral losses in the MS2 spectrum and runs MS3 measurements.
  • Dedicated software provides powerful support for the structural analysis of sugar chains.