Uncovering glycopeptide structural isomerism by mass spectrometry

Glycosylation is a widespread post translational modification essential to biological systems. Its structural diversity, arising from varied linkages, branching patterns, and monosaccharide compositions, creates a need to distinguish glycan composition from glycan structure. This thesis advances structural identification and relative quantification of glycopeptide isomers using LC MS/MS–based approaches. Chapter 1 introduces glycosylation fundamentals, including glycan building blocks and branching architectures, followed by an overview of tandem mass spectrometry. Ionization methods are discussed with emphasis on electrospray ionization, along with quadrupole isolation, fragmentation principles, and ion detection. The chapter compares data dependent and data independent acquisition strategies and concludes with a brief introduction to ion mobility–mass spectrometry, a technique increasingly used for structural glycan analysis. Chapter 2 reviews MS based strategies for glycan structural elucidation, focusing on gas phase methods such as fragmentation and ion mobility rather than chromatographic or derivatization based approaches. In glycomics, structural analysis has benefited from ion mobility and derivatization, whereas in glycoproteomics these tools remain underutilized due to technical challenges. As a result, structural glycoproteomics has relied on diagnostic ions and ion ratio based methods. Continued developments are expanding possibilities for structural analysis. Chapter 3 presents an MS2 based workflow for determining glycopeptide isomer ratios. Using isomer defined standards, distinct fragmentation behaviors were observed under collision energy gradients. Branching isomers showed higher low energy fragment abundance from the α1,3 linked mannose branch, while sialic acid linkage isomers exhibited higher medium energy NeuAc fragments for α2,3 linked species. These trends were converted into quantitative variables enabling relative isomer quantification. However, precursor charge state dependence limited applicability to small peptides where peptide influence is minimal. Chapter 4 incorporates HILIC separation to overcome peptide and charge state related limitations. Chromatographic resolution of isomers enabled MS2 to be used solely for structural identification, while quantification relied on peak areas. This approach revealed subclass specific IgG glycosylation signatures: IgG1 and IgG3 predominantly galactosylate the α1,6 antenna, IgG2 favors the α1,3 antenna, and IgG4 shows a balanced pattern. These features were consistent across recombinant and endogenous IgGs, uncovering structural differences relevant to therapeutic antibodies. Chapter 5 introduces an MS3 workflow designed to eliminate peptide and charge state effects on glycan fragmentation. This method enabled determination of GlcNAc linkage in N5H6S3 glycans and NeuAc linkage ratios across sialylated glycopeptides. Analysis of B4GALT1, MGAT2, MAN1B1, COG5, and COG6 deficiencies revealed both compositional and structural abnormalities, including altered NeuAc linkage patterns, atypical GlcNAc positioning, and reduced α2,3 sialylation. This MS3 based approach is among the first capable of extracting structural glycan information from complex glycoproteomic samples. Chapter 6 discusses remaining challenges, including reliance on custom data analysis scripts, persistent charge state and peptide effects, and the need for isomerically defined standards. Developing generalized fragmentation rules and improving control of collision energy normalization may help address these limitations and support the maturation of structural glycoproteomics.