Antibody Glycosylation by Mass Spectrometry

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Antibodies (Abs) are glycoproteins of prime importance in the immune system, with immunoglobulin G (IgG) as their most common representative. There are many sources of IgGs and several motives to characterize their glycosylation patterns and amino acid (AA) sequences, which is the central theme of this proposal. The PI's laboratory has specialized in glycosylation analysis for over 20 years, and has trained many HQP. This proposal involves a minimum of 10 HQP over 5 years and each of them will benefit of interdisciplinary training in bioanalytical chemistry and mass spectrometry (MS). Sections of the proposal are summarized below.*******Part of IgG glycosylation occurs in the antigen-binding region (Fab domain) of the molecule, which varies from one type of IgG to the next with respect to AA sequence. As very little information is available in the literature about the variable portions of Fab domains, there is a strong need for the development of reliable methods and workflows to characterize the Fab with respect to glycosylation and AAs. This is important in order to learn more about Ab-antigen interactions and about the role of glycosylation in this process.*The IgGs will be from three sources: human plasma, swine plasma, and cell cultures from collaborators' laboratories.*******Human serum IgG is polyclonal, i.e. it has subtypes differing in their AA sequences (IgG1, IgG2, IgG3 and IgG4). Each subtype shows different levels affinity for specific receptors, but so far with MS it has been difficult to quantify these subtypes. It is proposed to develop a quantitative method which will use synthetic peptide standards unique to the sequence of each subtype. The ratios of these four subtypes has been found to vary slightly in the population, and this method will help investigate if immune system response may relate to these ratios.*******Also of interest are porcine IgGs. Differences in glycosylation between swine and human result in problems in the context of xenotransplantation (transplantation from animal to human). Normal pig organs are rejected by humans, and this is partly attributed to specific terminal sugars on the proteins. By knocking out (KO) the genes responsible for these, human-like organs and fluids may be generated for xenotransplantation. The objective is to fully characterize IgG glycosylation, in order to assess the efficiency of the KO process and understand the role of glycans in the rejection phenomena.*******IgG glycosylation analyses will be conducted using glycoproteomic approaches, for which novel capture-release methods (cleavable linkers) are presently being investigated in the PI's laboratory for their ability to capture glycopeptides and tested on monoclonal Abs from bioreactors. These new materials consist of linkers synthesized onto solid phase materials, e.g. functionalized silica or magnetic nanoparticles. These products will be of great interest to the glycoproteomics scientific community.******