Complex network of sialylated glycans analyzed by LC-MS

Unlock Glycan Secrets: A New Way to Analyze Sialylated Structures

Nico Varela in Science & Nature September 2025 • 4 min read.

"Revolutionary LC-MS method provides deeper insights into glycan linkages, essential for understanding diseases and developing new treatments."

Glycans, complex sugar molecules found on the surface of cells, play vital roles in biological functions ranging from cell-to-cell communication to immune responses. Among these, sialylated glycans, which contain sialic acids (Sia), are particularly important. Sialic acids often terminate glycan chains and are linked in different ways (α2,3- or α2,6-linkages), creating structural diversity that influences their biological activity.

Understanding the precise structure of these sialylated glycans, including the specific linkages of sialic acids, is crucial for deciphering their functions in both normal and diseased states. For example, the linkage of sialic acids can determine the species tropism of influenza viruses. Avian influenza viruses preferentially bind to α2,3-linked Sia, whereas human influenza viruses favor α2,6-linked Sia. This specificity underscores the importance of accurate glycan analysis.

Traditional methods for analyzing glycans often fall short when it comes to distinguishing between these critical linkages. To address this challenge, researchers have developed a new method that combines liquid chromatography-mass spectrometry (LC-MS) with a technique called linkage-specific alkylamidation. This innovative approach allows for a more detailed and quantitative analysis of sialylated glycans, opening new avenues for research and potential therapeutic development.

What is Linkage-Specific Alkylamidation and How Does It Work?

Complex network of sialylated glycans analyzed by LC-MS

The core of this new method lies in a two-step chemical modification process called alkylamidation. This process specifically targets the carboxyl groups on sialic acids, modifying them in a way that reveals their linkage. Here’s a breakdown of the steps:

The process allows the carboxyl groups of α2,3- and α2,6-linked Sia to be derivatized with two kinds of alkylamines, which have different mass values, in a linkage-specific manner.

Step 1: The glycans are reacted with isopropylamine (iPA). This causes α2,6-linked Sia to form alkylamides. Meanwhile, α2,3-linked Sia will form lactones with neighboring galactose molecules.
Step 2: The lactones formed in the first step are then reacted with methylamine (MA). This converts the lactones into stable amide forms.

By using two different alkylamines, the researchers can distinguish between the two types of sialic acid linkages through mass spectrometry. The resulting mass differences allow for quantitative analysis, providing insights into the relative abundance of each linkage type within a sample.

Why This Matters

The development of this improved method for glycan analysis has significant implications for a wide range of biological and medical fields. By enabling more accurate and detailed characterization of sialylated glycans, researchers can gain a better understanding of their roles in various processes, from immune regulation to viral infections. This knowledge can then be leveraged to develop new diagnostic tools, therapeutic interventions, and even preventative strategies for diseases influenced by glycan structures.

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Everything You Need To Know

What are glycans and why is it important to study them?

Glycans are complex sugar molecules found on the surface of cells. They are critical in many biological functions, such as cell communication and immune responses. Sialylated glycans, which contain sialic acids, are particularly important because the specific way sialic acids link to the glycan chain influences their biological activity. Understanding glycan structures, especially sialic acid linkages, is vital for understanding diseases and developing new treatments. The methods provide a new way to analyze these structures with unprecedented detail.

What is linkage-specific alkylamidation and how does it work to analyze sialylated glycans?

Linkage-specific alkylamidation is a chemical process used to analyze sialylated glycans. It involves a two-step chemical modification that targets the carboxyl groups on sialic acids. In the first step, glycans react with isopropylamine, causing α2,6-linked sialic acids to form alkylamides, while α2,3-linked sialic acids form lactones. In the second step, these lactones react with methylamine to form stable amide forms. This process allows researchers to distinguish between α2,3- and α2,6-linked sialic acids through mass spectrometry, enabling quantitative analysis of each linkage type.

Why is it important to distinguish between α2,3- and α2,6-linkages of sialic acids?

The ability to differentiate between α2,3- and α2,6-linkages of sialic acids is crucial because these linkages determine the biological activity of sialylated glycans. For example, influenza viruses show a preference for specific sialic acid linkages: avian influenza viruses bind preferentially to α2,3-linked sialic acids, while human influenza viruses favor α2,6-linked sialic acids. Understanding these preferences is vital for studying viral infections, developing effective treatments, and implementing preventative strategies.

What is LC-MS/MS, and how is it used in the analysis of glycans?

LC-MS/MS, or liquid chromatography-mass spectrometry, is an analytical technique used to identify and quantify different molecules in a sample. In the context of glycan analysis, LC-MS/MS is used to separate glycans based on their physical properties using liquid chromatography, and then to determine their mass and structure using mass spectrometry. When combined with linkage-specific alkylamidation, LC-MS/MS allows for detailed and quantitative analysis of sialylated glycans, providing insights into the abundance of different sialic acid linkages.

What are the potential implications of using this method for glycan analysis in biological and medical fields?

This novel method improves the characterization of sialylated glycans, which helps researchers better understand their roles in various biological processes, such as immune regulation and viral infections. This knowledge can lead to new diagnostic tools, therapeutic interventions, and preventative strategies for diseases influenced by glycan structures. Additionally, by providing a more accurate and detailed analysis of glycan structures, this method can facilitate the development of targeted therapies that specifically interact with or modify these structures.