Determination of Sialic Acid Isomers from Released N-Glycans Using Ion Mobility Spectrometry

Complex carbohydrates are ubiquitous in nature and represent one of the major classes of biopolymers. They can exhibit highly diverse structures with multiple branched sites as well as a complex regio-and stereochemistry. A common way to analytically address this complexity is liquid chromatography (LC) in combination with mass spectrometry (MS). However, MS-based detection often does not provide sufficient information to distinguish glycan isomers. Ion mobility mass spectrometry (IM-MS) -a technique that separates ions based on their size, charge, and shape -has recently shown great potential to solve this problem by identifying characteristic isomeric glycan features such as the sialylation and fucosylation pattern. However, while both LC-MS and IM-MS have clearly proven their individual capabilities for glycan analysis, attempts to combine both methods into a consistent workflow are lacking. Here, we close this gap and combine hydrophilic interaction liquid chromatography (HILIC) with IM-MS to analyze the glycan structures released from human alpha-1-acid glycoprotein (hAGP). HILIC separates the crude mixture of highly sialylated multi-antennary glycans, MS provides information on glycan composition, and IMS is used to distinguish and quantify alpha 2,6-and alpha 2,3-linked sialic acid isomers based on characteristic fragments. Further, the technique can support the assignment of antenna fucosylation. This feature mapping can confidently assign glycan isomers with multiple sialic acids within one LC-IM-MS run and is fully compatible with existing workflows for N-glycan analysis.

Automated summary

Complex carbohydrates are common in nature and can have diverse structures, making their analysis challenging. Liquid chromatography combined with mass spectrometry is a common method for addressing this complexity, but ion mobility mass spectrometry has shown potential for distinguishing glycan isomers. Combining hydrophilic interaction liquid chromatography with IM-MS can be used to analyze glycan structures released from proteins, providing information on glycan composition and allowing for the distinction and quantification of glycan isomers with multiple sialic acids.