Fc Profiling of Polyclonal IgG, IgA and IgM by Light Chain Capturing Coupled with NanoRP-LC-MS

The proteoform profile of antibody Fc domains determines
antibody
effector functions, not only for biopharmaceuticals but also for endogenous
antibodies. Endogenous immunoglobulin G (IgG) Fc-proteoforms have
been well characterized by using different MS-based approaches, comprising
bottom-up and intact Fc domain workflows. However, assessment of IgA1
and IgM Fc domains is still challenging, due to the more complex structure,
and analyses have been limited to the peptide level only. In this
work, a light-chain affinity capturing workflow combined with isotype-specific
hinge-region digestion and subsequent intact Fc domain nanoRP-LC-MS
analysis has been developed. The novel approach shows very good sensitivity
and precision, enabling simultaneous capturing of antibody isotypes
with sequential release and analysis of IgG, IgA1 and IgM Fcs from
10 μL of plasma. Single donor human samples were successfully
analyzed, providing a comprehensive overview on Fc proteoforms but
also on associated Fc-components such as the joining (J) chain of
IgA and IgM and CD5L.