Glycans have long been known to play significant roles in biological systems and are essential to all life forms. Complex and diverse glycans appear to be ubiquitous in all cells throughout nature. Glycomics emerged with the goal of defining the glycome of a biological system to better assess the biological attributes of the glycans, which is indispensable in the comprehensive and systematic understanding of living organisms. Thereinto, glycomic quantitation is an important part in glycomics, which is essential for identifying the differential expression of glycans in diseases and biological processes. labeling by stable isotope compound is still the main approach in glycomic quantitation, and now has developed a variety of stable isotope (D, 13C, 15N, and 18O) labeling strategies that include live cells or organisms (metabolic in vivo labeling) and extracts or purified glycans (chemical or enzymatic in vitro labeling).

GlycomicsFig. 1 The schematic diagram of isotopic labeling for glycomic quantitation.

Advantages of stable isotope labeling


Compared with label-free methods, isotope labeling techniques have the advantage that a stable isotope-labeled internal standard can be used to compensate for changes in instrument performance over time and have high quantitative accuracy in which the glycomic quantitative ability is not easily influenced by the experimental operation and stability of the analyzer.

Labeling strategy

For the labeling of glycans by isotope-labeled compound, three main isotope labeling strategies including, chemical isotope labeling, enzymatic isotope labeling and metabolic isotope labeling have been applied [1].

Chemical isotope labeling

Chemical isotope labeling of glycans has been continuously developed and implemented for quantitative glycomics. The most popular method is using isotope-labeled iodomethane, or other labeled reagents labeling glycans by reductive amination or NHS-ester reactions to form labeled permethylated glycans. Then the unlabeled and labeled glycans that have been permethylated are compared. The isotope-labeled iodomethane includes CH2DI, CHD2I, CD3I, 13CH3I, 13CH2DI, 13CHD2I, 13CD3I, and CH3I, and their reagent pairs (12CH3I/13CH3I, CH3I/CH2DI, CH3I/CD3I). Other labeled reagents are main the three stable isotopes, 13-carbon, deuterium, and 15-nitrogen such as 13C6-aniline, 13C6-2-aminobenzoic acid, 13C6-2-aminobenzoic acid, D4-2-aminobenzoic acid and 13C6-phenethylbenzohydrazide.

GlycomicsFig. 2 Permethylation of glycans by isotopically labeled iodomethane.

Enzymatic isotope labeling

The enzymatic isotope labeling of glycans was developed mainly using the mechanism in which 18O is incorporated into the reducing end of N-linked glycans through hydrolysis of endoglycosidase in the presence of H218O water. Other acceptors than water also can be used in transglycosylation reactions in enzymatic isotope labeling. As shown in Fig. 3, a novel relative quantification method of N-linked glycans by utilizing transglycosylation with Endo-M (a glycoside hydrolase (GH) family 85 enzyme) and stable isotope-labeled glucose (U-13C6) as an acceptor.

GlycomicsFig. 3 In vitro enzymatic isotope labeling strategies by isotope-labeled acceptor and transglycosylation of Endo-M.

Metabolic isotope labeling

In addition to the chemical and enzymatic approaches above, metabolic isotope labeling strategies of glycans with isotope-labeled monomer building blocks (glutamine or glucose) have been introduced as viable alternatives. Generally, metabolic isotope labeling of glycans using 15N isotope-labeled glutamate and 13C isotope-labeled glucose in cell culture.

What can we do?

Alfa Chemistry has many years of experience in the synthesis and development of stable isotope-labeled products. We can provide various stable isotope-labeled compounds for glycomic quantitation use, including 13-carbon, deuterium, and 15-nitrogen labeled iodomethane, glutamate, glucose, etc. And our professional technology teams that can also provide customers with high-quality stable isotope-labeled compounds design and customization services. No matter what design ideas you have, we will implement them together with you. We aim to apply various isotope labeling method into quantitative glycomics and then extend to studies of glycobiology and clinical investigations. Please contact us immediately to order or cooperate in research and development with high quality and reasonable price.


  • Yun, J., et al. Isotope labeling strategies of glycans for mass spectrometry-based quantitative glycomics[J]. Microchemical Journal, 2021, 170: 106655.
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