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

Abstract

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 α2,6- and α2,3-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.

Amide and Multihydroxyl Complementary Tailored Metal–Organic Framework with Enhanced Glycan Affinity for Efficient Glycoproteomic Analysis

“…Figure e illustrates the N -glycan structures with the top 15 relative abundances identified in both CTR and T2D human serum. Notably, elevated levels of complex-type acidic N -glycan, such as [Hex]­5­[HexNAc]­4­[NeuAc]­2, were significantly observed in T2D, which is commonly associated with cancer and characterized by terminal N -acetylglucosamine . In contrast, neutral glycans such as [Hex]­4­[HexNAc]­4­[Fuc]­1 were highly expressed in the CTR group, which displayed marked differences between the CTR and T2D groups.…”

“…Notably, elevated levels of complex-type acidic N-glycan, such as [Hex]5[HexNAc]4[NeuAc]2, were significantly observed in T2D, which is commonly associated with cancer and characterized by terminal N-acetylglucosamine. 37 In contrast, neutral glycans such as [Hex]4[HexNAc]4[Fuc]1 were highly expressed in the CTR group, 38 which displayed marked differences between the CTR and T2D groups. It is noteworthy that U6N/Pv@Glc facilitated the enrichment and identification of numerous low-abundance neutral glycans (excluding the top 5) bearing multiple fucose (deoxyhexose) residues, which was attributed to the advantageous capability of amide-HILIC in effectively separating neutrally charged glycans.…”

Ion Mobility-Tandem Mass Spectrometry of Mucin-type O-Glycans

“…Recent advancements in ion mobility technology highlighted the potential of the method to be used in LC-based glycomics analyses. 23,[37][38][39][40][41][42] Fundamental studies recently showed that IMS can be theoretically described using a plate-height model, similarly to other separation techniques such as capillary zone electrophoresis or LC. 43,44 Dedicated bioinformatics tools for deconvolution and data treatment further strengthen the utility of ion mobility in real-world applications.…”

Essential functions, syntheses and detection of sialyl Lewis X on glycoproteins

“…With the development of technology, more and more detection approaches have emerged. For instance, ion mobility spectrometry (IMS) has been shown to separate and identify α2-3 and α2-6 linked Neu5Ac from released N-glycans [141]. IMS possesses an essential dimension which is able to resolve isobaric species by their gas-phase collision cross-section (CCS), however, the application of IMS is usually limited by the databases of carbohydrate CCS values [142,143].…”