The Study of N-glycans using HILIC, Ion Mobility, and Mass Spectrometry

N-linked glycosylation is an important post translational modification, and the changes in N-glycan patterns are known to be associated with various human diseases. The study of N-glycans is crucial for the safety and efficacy of biotherapeutics. Tandem mass spectrometry (MS/MS) is a popular method in glycomics where glycans are identified via their mass to charge (m/z) and fragment ions. However, glycans exist as isomers arising from linkage, anomeric, and stereochemical differences. These isomers are difficult/impossible to differentiate using only MS approaches. Hydrophilic interaction liquid chromatography (HILIC) is a popular LC mode that complements mass spectrometry in glycomics study. For instance, HILIC can distinguish α-2,6 sialylated from α-2,3 sialylated glycans, which are challenging linkage isomers to identify by mass spectrometry. HILIC-MS/MS has been demonstrated as a powerful tool and can characterize N-glycans in different complex matrices including cells or monoclonal antibodies. Recently, HILIC-MS/MS was used for the detection of immunogenic α-Gal glycans in the presence of their isomeric species. However, this system struggles to detect some α-Gal isomers, which suggests the need for improvement. High-resolution Ion Mobility Spectrometry (IMS) was added to increase the resolving power of the HILIC-MS system. The improved separations provided by the HILIC-IMS-MS system has enabled the identification of numerous isomeric N-glycans including those containing α-Gal from their non-α-Gal isomers. The study of N-glycans was carried on using HILIC-IMS-MS and revealed some unseen isomers, indicating the power of multidimensional HILIC-IMS-MS system in identifying highly complex glycans.  Drift time fingerprints were collected from IMS for each glycan species that can be used as an additional dataset to pair with retention time collected from HILIC, and m/z data from MS. The 3-D dataset significantly increases the efficiency and accuracy in identifying complex isomers for high throughput data analysis.