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Tissue N-Glycan Analysis Using LC-MS, MS/MS, and MSn
1Graduate School of Science and Technology, Niigata University, Niigata, Japan.
Current Protocols
|July 27, 2021
Summary
This study presents a detailed protocol for analyzing complex tissue N-glycans. 2-Aminopyridine tagging and LC-MS/MS enable detailed structural elucidation of these important biomolecules.
Area of Science:
- Glycomics and Mass Spectrometry
- Biochemistry and Molecular Biology
- Analytical Chemistry
Background:
- Tissue N-glycans exhibit significant structural heterogeneity, impacting biological functions.
- Accurate and comprehensive analysis of N-glycan structures is crucial for understanding biological processes and disease states.
- Existing methods often struggle with the complexity and low abundance of certain glycan structures.
Purpose of the Study:
- To provide detailed protocols for the comprehensive structural analysis of N-glycans released from tissue samples.
- To establish a robust method for isomer separation and quantitative analysis of both major and minor N-glycan structures.
- To enable detailed structural elucidation, including core structures, branching patterns, and glycosidic linkages.
Main Methods:
- Release of N-glycans from glycoproteins/glycopeptides using glycoamidase F or hydrazinolysis.
- 2-Aminopyridine (PA) tagging of released N-glycans for enhanced separation and detection.
- Reversed-phase liquid chromatography (LC)-mass spectrometry (MS) and MS/MS analysis, complemented by anion-exchange chromatography, exoglycosidase digestions, and sialic acid linkage-specific alkylamidation (SALSA) with optional permethylation.
Main Results:
- PA-N-glycans allow for effective separation of isomers and quantitative analysis of diverse glycan structures.
- Retention behavior on reversed-phase C18 columns aids in deducing core structures (e.g., core Fuc, bisecting GlcNAc) and branching patterns.
- Combined techniques provide detailed information on glycosidic linkages, particularly for sialic acids.
Conclusions:
- The presented protocols offer a powerful and versatile approach for in-depth N-glycan profiling of tissues.
- This methodology facilitates the identification and quantification of complex and isomeric N-glycans, advancing glycomic research.
- The detailed structural information obtained is vital for understanding the biological roles of glycans in health and disease.
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