SmartEnzymes in Targeted Sequencing of Heavily Glycosylated IgA1

The use of IgG-based antibodies in various clinical fields have increased over that past decades, continuing the development of better biopharmaceuticals. The use of other immunoglobulins including IgA, characterized with the ability to recruit effector cells, is progressively being considered a useful alternative. The complexity of the heavily glycosylated IgA does however pose analytical challenges and no method currently exist that allows unraveling of the human repertoire of this subclass.


Scientists at Utrecht University present a mass spectrometry method using electron capture dissociation (EDC) to obtain sequence ladders of the variable regions on the heavily N- and O-glycosylated anti-CD20 IgA1 antibody. Using SmartEnzymes and a native top-down approach, the scientists compared the IgA1 antibody to its anti-CD20 IgG counterpart, and their Fabs.


To obtain intact Fab and Fc subunits, the IgG antibody was digested at a specific site above the hinge using the FabALACTICA enzyme. To tackle the heavy O-glycosylation of the IgA1 antibody, the scientists combined the SialEXO and OpeRATOR enzymes. Hydrolysis of sialic acids using SialEXO allowed for the OpeRATOR enzyme to digest the IgA1 glycoprotein N-terminally of core1 O-glycan sites located above-hinge.


Using the native top-down mass spectrometry method, the scientists were able to obtain straightforward-to-read sequence ladders of both the light and heavy chain variable parts of IgA1, and specifically the critical CDR3 region. The sequence data obtained in this work highlights the potential of native top-down ECD as a complementary method for de novo sequencing of IgA.


Greisch et al., 2021. Generating Informative Sequence Tags from Antigen-Binding Regions of Heavily Glycosylated IgA1 Antibodies by Native Top-Down Electron Capture Dissociation. Journal of the American Society for Mass Spectrometry. doi: 10.1021/jasms.0c00461



SialEXO – Sialidases for removal of sialic acids from glycoproteins.
OpeRATOR – O-glycan specific protease for mucin-type O-glycosylated proteins.
FabALACTICA – Cysteine protease for above hinge digestion of human IgG1.