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The development of top-down and middle-level analytical HPLC-MS strategies in recent years mainly focused on the characterization of therapeutic monoclonal antibodies. In our research, we aim at developing similar strategies to analyze polyclonal IgG with regard to subclass abundance and glycosylation patterns, which may provide new insights into immune regulatory processes. However, when dealing with polyclonal IgGs, we faced the challenge of pronounced molecular heterogeneity arising from sequence variability. In this context, we took advantage of the glycosylated Fc domain, which is conserved in polyclonal antibodies and determines the IgG subclass. In our recent work, we investigated the feasibility of middle-level analysis by FabULOUS (SpeB) digestion and HPLC-MS of polyclonal mouse IgG.
How did SmartEnzymes enhance your work?
The FabULOUS enzyme was exploited for the generation of Fc/2 subunits from all murine IgG subclasses, which all were proven amenable for proteolytic cleavage. The obtained subunits enabled the dissociation of Fc and Fab domains required to tackle the immense sequence heterogeneity within the variable regions. Middle-down analysis by HPLC-MS of the Fc/2 subunits allowed the assignment of the specific isotypes, while middle-up analysis provided quantitative information on the subclasses as well as their respective glycosylation variants. The workflow thereby enables global analysis of polyclonal murine IgGs with respect to subclass abundances including closely related isotypes as well as glycosylation profiles and other PTMs. Finally, we demonstrated the capabilities of our workflow in a pilot study dealing with polyclonal IgG from mouse serum after immunization with pollen allergen.
In summary, the described middle-level workflow provides comprehensive information obtained in a single analysis involving swift sample preparation, standard HPLC-MS analysis, and straightforward data evaluation as an attractive extension to the toolbox of analytical strategies for antibodies.