Proteomics > Which service should I request? > Glycan / Glycoprotein analysis > Characterization of N-linked glycans

Characterization of N-linked glycans


General description

Glycomics "is the systematic study of all glycan structures of a given cell type or organism" and is a subset of glycobiology. To characterize N-glycans, the protein mixtures would be first extracted from their biological resources. Then, the N-glycans are released by PNGase F or PNGase A depending on the species. To increase the sensitivity of glycans on MS-based detection, glycans would be further permethylated before MS analysis. The MS profile can provide an overview of N-glycome from samples. For advanced structure characterization, tandem mass spectrometry would be applied to confirm the linkage of glycan structures. For O-glycomics, please contact us for further information.



Based on the biological question and on the type of sample, a typical workflow for processing the samples prior to MS and data analyses consists of:

1. Protein digestion/ glycan release and permethylation

  • Protein digestion: Proteins mixtures extracted from different biological resources, for example, cells or biological fluid, are first digested into peptides.
  • Glycan release: For N-glycomic analysis, samples are treated with PNGase F/A. The released glycans would be purified by a reverse-phase cartridge.
  • Permethylation: Glycans are chemically methylated by methyl iodide.
The principle of permethylation is shown below. In addition, the fragmentation pattern of glycans in MS/MS analysis is shown in the lower panel.


2. MALDI-MS analysis

At FGCZ, the default data acquisition is operated by Bruker Ultraflextreme MALDI-TOF-TOF. Samples are dissolved in pure acetonitrile and mixed with MADLI matrix, 10 mg/mL of 2,5-dihydroxy-benzoic acid (DHB), and spotted on the MALDI target.

3. Data analysis

The data are processed by Flexanalysis (Bruker). The annotation is done by Glycoworkbench 2.0 and also manual inspection.

Created by chiawei. Last Modification: 2023-02-14 14:33 by paolo.