Direct comparison of the hinge-cleaving proteases IgdE and BdpK for LC-MS based IgG1 clonal profiling

ABSTRACT: Human antibodies are heterogeneous molecules, primarily due to clonal sequence variations. Analytical techniques to assess antibody levels quantitatively, like ELISA assays, lack the power to determine abundances at the clonal level. Recently, we introduced an LC-MS-based approach that can distinguish and quantify antibody clones using the mass and retention time of their corresponding Fab-fragments. We used a specific hinge-cleaving protease IgdE (FabALACTICA) to release the Fab-fragments from the glycosylated constant Fc region of the antibody. Here, we explore an alternative protease, the recently described IgG1-specific hinge-cleaving protease BdpK (FabDELLO) and compare it directly to IgdE for its applicability in IgG1 repertoire profiling. We used IgdE and BdpK in parallel to digest the IgG1s from the same set of plasma samples. Both proteases cleave IgG1 in the hinge region, albeit at two distinct cleavage sites. The LC-MS based analyses of the Fab-fragments generated by IgdE or BdpK produced qualitatively and quantitatively very similar clonal repertoires. However, IgdE required ~16 hours to digest the polyclonal plasma IgG1s, while BdpK required just ~2 hours. We validated the similarity of the clones by top-down proteomics, using Electron Transfer Dissociation (ETD). We conclude that BdpK performs well in digesting polyclonal plasma IgG1 samples, and that neither BdpK nor IgdE displays detectable biases in cleaving IgG1s. We anticipate that BdpK may emerge as the preferred protease for IgG1 hinge- digestion because it offers a shorter digestion time compared to IgdE, an equally specific digestion site, and no bias against any IgG1 present in the plasma repertoires.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Albert J.R. Heck  

PROVIDER: MSV000092676 | MassIVE | Thu Aug 17 00:25:00 BST 2023

REPOSITORIES: MassIVE