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Abstract
The existence of different isomers makes the study of N-glycan a challenging task. Hydrophilic Interaction Liquid Chromatography paired with Mass Spectrometry (HILIC-MS) is one of the well-known methods in glycomics. Having a single separation dimension limits HILIC-MS ability to distinguish some isomers. Ion mobility spectrometry (IMS) is a high resolution gas-phase separational technique that can be combined with HILIC-MS to increase the resolving power of the system without compromising the efficiency of HILIC separation. The HILIC-IMS-MS system provides better separation for N-glycan isomers, improving the ability to identify individual components in complex carbohydrate mixtures. The work presented in this dissertation highlights the application of HILIC, IMS, and MS for the study of N-glycans. In Chapter 3, HILIC-MS was employed to examine the quantitative results of N-glycans labeled with different tags. The quantification of N-glycans using liquid chromatography (LC) – MS was found to be impacted by the chosen derivative tag. The ability of HILIC-MS/MS to distinguish glycan isomers was evaluated in Chapter 4. Separations were achieved between linkage and branched isomers. Chapter 5 describes the analysis of immunogenic N-glycans containing α-linked Galactose (α-Gal), HILIC-MS/MS could not separate α-Gal glycan from its non-α-Gal isomer. IMS successfully enabled the separation between α-Gal glycan and its non-α-Gal isomer. Because the high resolving power of IMS complements the performance of HILIC-MS for isomeric separation, a database (Chapter 6) was created that includes HILIC retention time, ion mobility collision cross section (CCS), and mass to charge (m/z) data of 205 procainamide labeled N-glycans. The database acts as a resource for data analysis of unknown samples.
An orthogonality study was performed to evaluate the resolving power obtained by combining different LC modes of separations with IMS-MS (Chapter 7). For glycan, porous graphitic carbon (PGC)-IMS-MS has higher peak capacity compared to HILIC-IMS-MS. Similar study performed on peptide shows that HILIC-IMS-MS has higher peak capacity than reserved phase (RP)-IMS-MS.