Glycoqueuing: Isomer-Specific Quantification for Sialylation-Focused Glycomics

Abstract

Changes of α-2,3-/α-2,6-linked sialic acids (SAs) in sialylglycans have been found to be closely related with some diseases. However, accurate quantification of sialylglycans at the isomeric level remains challenging due to their instability, structural complexity, and low mass spectrometry (MS) detection sensitivity. Herein, we propose an analytical strategy named "glycoqueuing", which allows sequential chromatographic elution and high-sensitivity MS quantification of various sialylglycan isomers based on isotopic labeling followed by analysis via online reversed-phase high performance liquid chromatography coupling with MS (RP-HPLC-MS). The new method was validated by detailed structural identification and quantification of fetal bovine serum (FBS) N-linked sialylglycan isomers, during which many branching isomers were successfully differentiated, and 28 sialylglycan compositions with Neu5Gc residues were analyzed. The method was successfully applied to isomer-specific, quantitative comparison of sialylated N-glycans between bovine and rabbit immunoglobulin G (IgG) and the search for serum sialylated N-glycan biomarker candidates of hepatocellular carcinoma, during which a 55% increase of α-2,6-sialylated fucosylated N-glycans was revealed, demonstrating the great applicability and potential clinical usage of the method.

Glycomics and glycoproteomics: Approaches to address isomeric separation of glycans and glycopeptides

“…used a long C18 column to investigate the heterogeneity of SARS‐CoV‐2 S protein [86]. Since RP columns exhibit relatively good reproducibility and stability, researchers work on the alkyl columns to separate isomers like standard N‐ glycans, O‐ glycans, oligosaccharides, or less complex structures [79,83,87,88].…”

“…Moreover, the reaction selectivity for the α2,3 and α2,6 sialic acid linkages is used to differentiate between isomeric‐linked structures. The selective reaction of α2,3 linked sialic acids undergo the formation of lactones with the adjacent sugars of the glycan residues, and the α2,6 linked sialic acids undergo the carboxylic acid derivatization into amides or esters, differences that can be easily observed in the product molecular masses [87]. Otherwise, the nonselective reactions convert the carboxylic acids of both sialic acid linkages into amides or esters in one step, avoiding the formation of labile lactones for the α2,3 linkages.…”

“…Additionally, a strategy to prevent sialic acid loss and increase the ionization efficiency and isomeric separation of sialylated glycans was developed by Jin et al., who used GP as the reducing end label and derivatized the sialic acid moieties with aniline. The results showed a total of 41 isomeric structures resolved from 17 glycan compositions and demonstrated that 28 glycan structures have N‐ glycolylneuraminic acid (Neu5Gc), which are difficult to see due to their low abundance [87]. Other labels that have proved to be effective for the separation of isomeric glycans are 8‐animonaphthalene‐1,3,6‐trisulfonic acid (ANTS) [83,136] and 2‐aminopyridine [137].…”

“…Thus, in order to have reliable alternatives for the identification of glycans and glycopeptides, several approaches have been reported. Recently, some researchers have suggested using the retention time and structural hydrophobicity in LC‐MS analysis to increase evidence of the presence of glycans and glycopeptides, and to complement the MS n fragmentation [87,145,148–150]. A promising alternative published by Jin and co‐workers, called “glycoqueuing,” was used to separate sialylated isomeric glycans from human and bovine serum samples.…”

“…The research group showed that various isomeric glycan structures, derived from the presence of the sialic acid in different antennas or with different linkages, produced an elution pattern related to the hydrophobicity and the sialic acid linkage. Therefore, those differences and their direct relation with the RT can be used for the identification of sialylated isomeric glycan structures (Figure 7A‐C) [87]. Later, Klein and Zaia proposed a relative retention time estimation tool that can improve the identification of N‐ glycopeptides analyzed by LC‐MS.…”