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Sample clean-up method for analysis of complex-type N-glycans released from glycopeptides
Miyako Nakano1, Kazuaki Kakehi, Yuan C Lee
1Faculty of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka 577-8502, Japan.
Journal of Chromatography. A
|August 20, 2003
Summary
Liberating N-glycans from glycopeptides is efficient, but requires removing interfering peptides. A novel method using sodium 2,4,6-trinitro-benzene-1-sulfonate (TNBS) and graphitized carbon effectively purifies N-glycans for analysis.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Glycobiology
Background:
- N-glycans are crucial for glycoprotein function.
- Liberating N-glycans from glycopeptides offers advantages over using intact glycoproteins.
- Peptidic contaminants interfere with N-glycan analysis and labeling.
Purpose of the Study:
- To develop an effective method for removing interfering peptidic materials from N-glycans.
- To enable high-performance anion-exchange chromatography (HPAEC) analysis of N-glycans.
- To improve the labeling of N-glycans by reductive amination.
Main Methods:
- Enzymatic liberation of N-glycans from glycopeptides using glycoamidases.
- Modification of peptide amino groups with sodium 2,4,6-trinitro-benzene-1-sulfonate (TNBS).
- Purification using graphitized carbon cartridges and octadecyl (C18) silica cartridges.
Main Results:
- Standard methods failed to completely remove interfering peptidic materials.
- TNBS modification increased peptide hydrophobicity, enhancing retention on cartridges.
- A combination of TNBS reaction and graphitized carbon treatment successfully removed interfering materials for HPAEC analysis.
Conclusions:
- The TNBS modification combined with graphitized carbon treatment is a highly effective strategy for purifying N-glycans.
- This method overcomes limitations of previous purification techniques.
- It facilitates accurate N-glycan analysis and subsequent labeling.