posted on 2023-12-18, 07:03authored byDanique
M. H. van Rijswijck, Albert Bondt, Naomi de Kat, Rolf Lood, Albert J. R. Heck
Human antibodies
are heterogeneous molecules primarily due to clonal
sequence variations. Analytical techniques to assess antibody levels
quantitatively, such as ELISA, lack the power to resolve 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 specific
hinge-cleaving protease IgdE (FabALACTICA) to release the Fab-fragments
from the constant Fc region of the antibody. Here, we explore an alternative
IgG1 hinge-cleaving protease, BdpK (FabDELLO), and compare it directly
to IgdE for use in IgG1 repertoire profiling. We used IgdE and BdpK
in parallel to digest all IgG1s from the same set of plasma samples.
Both proteases cleave IgG1 specifically in the hinge, albeit via different
mechanisms and at two distinct cleavage sites. Notwithstanding these
differences, the Fab fragments generated by IgdE or BdpK produced
highly similar clonal repertoires. However, IgdE required ∼16
h of incubation to digest plasma IgG1s, while BdpK required ∼2
h. We authenticated the similarity of the clones by top-down proteomics
using electron transfer dissociation. We conclude that BdpK performs
very well in digesting polyclonal plasma IgG1s 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 plasma.