Ozgumus Nitride, Beste (2024) Development of an In-Depth N-Glycan Profiling Workflow. [Tesi di dottorato]

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Abstract

This study focuses on the development of tandem mass spectrometry (MS/MS) workflows for the analysis of N-glycans, specifically those labeled with RapiFluor-MS (RF-MS), using recombinant immunoglobulin G1s (IgG1s) and glycoprotein hormones as model glycoproteins. The initial objective is to enhance the signal quality and acquisition rate of MS/MS spectra by optimizing key instrumental parameters such as spray voltage, radio frequency (RF) lens, and Automatic Gain Control (AGC) target. These optimizations have led to significant improvements in signal intensity and fragmentation efficiency, particularly in higher-energy collision dissociation (HCD) spectra, which are ideal for N-glycan sequencing. After optimizing the instrumental parameters, N-glycan fragmentation has been studied more in detail with a specific focus on triggering cross-ring fragments. A native FA2 standard has been used to better evaluate the effect of reducing end labeling on fragmentation. Fragmentation studies of labeled and native N-glycans using various dissociation techniques, including HCD, electron-transfer dissociation (ETD), and electron-transfer and higher-energy collision dissociation (EThcD), revealed distinct fragmentation patterns. While HCD spectra yielded abundant glycosidic fragments for protonated adducts, which facilitated comprehensive N-glycan composition determination, ETD and EThcD spectra were less efficient, particularly for labeled glycans. Substantial cross-ring fragments were obtained with sodium adducts nonetheless, they were limited to the reducing end or labeled end therefore have limited utility. After an insight of RF-MS labeled N-glycan fragmentation, LC-MS/MS workflow was developed to map the N-glycan profiles of two recombinant IgG1s both possessing unique glycosylation patterns. The workflow was optimized to improve chromatographic separation of isomeric N-glycans and to increase the acquisition rate of tandem MS spectra. The detailed MS/MS analysis revealed the presence of several rare N-glycan species in one of the IgG1s which were characterized for the first time. Notably, the workflow enabled the identification of isomeric N-glycans with minimal mass differences, showcasing its sensitivity and precision. The developed workflow was further optimized for a recombinant glycoprotein with a highly acidic N-glycosylation profile. New structures were identified, some of which were very low abundant, highlighting the sensitivity of the optimized workflow. Careful observations have led to a set of rules and by virtue of these rules rare structures were identified. Finally, rapid monitoring LC-MS/MS workflows for N-glycans were developed using a library of identified signature ions. The first workflow uses unique signature ions in a narrow range to monitor specific structures. The second workflow utilizes relative intensity changes in common signals to monitor α-galactosylated, outer arm fucosylated, and high mannose N-glycans, despite their lack of unique signature ions. The reliability of both methods is reinforced through enzymatic cleavage when possible. Overall, these workflows simplify sample preparation and data interpretation, making them accessible for less experienced operators and improving throughput for glycan profiling in various research contexts.

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