N-Glycan Analysis Service

N-Glycan Analysis Service

Service Details


Protein glycosylation, the addition of monosaccharide or oligosaccharide chains (glycans) to peptide backbones, including proteins, lipids, or other organic molecules, is a common post-translational modification (PTM) that confers various biological function. Glycosylation analysis is challenging due to the complexity and isobaric nature of linked glycans.

N-glycosylation, in which the glycan is attached to the nitrogen side chain of asparagine, is the most widely known form of glycosylation in biotherapeutics. N-glycans have been reported to regulate a variety of biological processes, such as ligand-receptor interactions, immune responses, protein secretion and transport, and plays an important role in a variety of diseases. Therefore, analyzing the link between N-glycan structure and its function is an important research area in elucidating molecular mechanisms involved in pathogenesis, monitoring diagnosis, prognosis, and biopharmaceutical development and quality control.

Fig. 1. Examples of N-glycans found in the commonly used expression systems.Fig. 1. Examples of N-glycans found in the commonly used expression systems. (Zhang L, et al., 2016)

Our Services

LC-MS-based N-glycan analysis is now the mainstream analysis method. Creative Proteomics provides the following N-glycan analysis services to analyze glycans and their species, including but not limited to.

Experiment Process

Fig. 2. Workflow for N-glycan profiling service.Fig. 2. Workflow for N-glycan profiling service.

N-Glycans Analysis by MALDI-TOF MS

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a new type of ionization biological mass spectrometry developed in recent years. It is suitable for high-throughput screening of proteins or other biological macromolecules with high sensitivity for the structural characterization of glycosylated compounds. After the N-glycans of the sample are released using N-glycosidase A or F enzymatic reactions, the released glycans can be rapidly analyzed by MALDI-TOF MS, where neutral and sialylated glycans are analyzed simultaneously in positive ion mode. We offer the following two analysis methods.

Permethylated N-Glycans Analysia by MALDI-TOF MS

Glycan permethylation with methyl iodide was used to improve ionization efficiency and stabilize sialylated glycans, then N-glycans were detected by MALDI-TOF MS.

  • Protein denaturation.
  • Enzymatic removal of N-glycans.
  • Labeling (permethylated).
  • Glycan analysis by MALDI-TOF MS.
  • Report.

Sialylated N-Glycans Analysis by MALDI-TOF MS

Stabilizes sialic acids by linker-specific sialic acid esterification, focusing on the differentiation of α-2,3 and α-2,6 sialic acid linkages and NeuNAc and NeuNGc residues in the N-glycan profile.

  • Protein denaturation.
  • Enzymatic removal of N-glycans (PNGAse F).
  • Enrichment of sialylated glycans.
  • Stabilization of sialic acid residues by esterification/lactonation.
  • N-glycan analysis by MALDI-TOF MS.
  • Report.

N-Glycan profiling by HILIC-UHPLC MS

Hydrophilic interaction chromatography (HILIC) can be used for N-glycan analysis. [4-amino-N-(2-diethylaminoethyl) benzamide]-labeled N-glycans (procainamide-labeled N-glycans) were analyzed by fluorescence and mass spectrometry detection. Compared with the traditional labeling methods of 2-aminobenzamide (2-AB) and 2-aminobenzoic acid (2-AA), we provide procainamide [4-amino-N-(2-diethylaminoethyl) benzamide]-labeled N-glycans show enhanced ESI ionization efficiency and fluorescence intensity, and thus improve identification of low abundance N-glycans.

  • Protein denaturation.
  • Enzymatic removal of N-glycans (PNGAse F).
  • Fluorescence labeling of N-glycans with procainamide.
  • Glycan analysis by HILIC-UHPLC MS.
  • Report.

*Note: Creative Proteomics can also perform glycan studies at the glycopeptide or glycoprotein level, providing you with complete N-glycan characterization services for glycosylation.

Advantages of Our N-Glycan Analysis Service

  • Excellent retention and powerful separation.
  • Advanced technology platform.
  • The analysis speed is fast, and the information is intuitive.
  • High throughput with sensitivity and specificity.
  • Stability and consistency without extensive re-testing.
  • Fully custom designed one-stop service.

ICH Q6B guidelines require comprehensive characterization of glycosylation of glycoproteins. With years of experience and an experienced scientific team, Creative Proteomics provides high-quality N-glycan analysis services. Additionally, we can provide fully custom project designs to meet any specific requirements. If you are interested, please contact us or send us an inquiry directly.


  1. Zhang L, Luo S, Zhang B. (2016) Glycan analysis of therapeutic glycoproteins. MAbs. 8(2):205-215.
  2. Kotsias M, Blanas A, van Vliet S J, et al. (2019) Method comparison for N-glycan profiling: Towards the standardization of glycoanalytical technologies for cell line analysis. PLoS One. 14(10):e0223270.

For research use only, not intended for any clinical use.

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